BG63565B1 - Method for changing the humoral immune response, method for the treatment,prevention or elimination of human immunodefficient virus in mammals and pharmaceutical compositions thereof - Google Patents

Abstract

The invention relates to compositions and methods including agents blocking the lymphotoxin- receptor signalization and affect immunological diseases, in particular antibody mediated immune responses. 31 claims, 7 figures

Description

This invention relates to compositions and methods comprising "lymphotoxin-β receptor blocking agents" that block lymphotoxin-β receptor signaling. Lymphotoxin-β receptor blocking agents are used to treat immunological diseases, in particular to suppress antibody-mediated immune responses, regulating the expression of addressins and cellular movement and affecting the differentiation of follicular dendritic cells. This invention relates to soluble forms of the lymphotoxin-β receptor extracellular domain and antibodies directed either against the lymphotoxin-β receptor or its ligand, surface lymphotoxin, which act as lymphotoxin-β receptor blocking agents.

BACKGROUND OF THE INVENTION

There are two types of acquired immunity that, when able to interact to achieve the overall goal of eliminating the antigen, are mediated by different elements of the immune system, with different effects. One type of acquired immune response, humoral immunity, is mediated mainly by B cells and circulating antibodies. The other type of cell or cell mediated immunity is mediated by T cells that synthesize and secrete cytokines that act on other cells.

Activation and differentiation of B cells in response to multiple antigens requires (1) the B cells to receive an antigenic signal through their antigen receptor-specific membrane Ig and (2) the cells to receive contact-dependent or independent signals from activated T-cells. The contact-dependent costimulatory signal results from the binding of the CD40 receptor on B cells to the CD40 ligand expressed on activated T-helper cells. (Laman et al., Crit Rev. Immunol., 16, pp. 59-108, (1996); Van Kooten and Banchereau, Adv. Immunol., 61, pp. 1-77 (1996). Contact-independent signaling is mediated from cytokines synthesized and secreted by activated T cells These contact-dependent and independent signals together induce B cells to differentiate either to (1) memory B cells, mediating a faster response upon secondary antigen entry or to ( 2) antibody secreting plasma cells Plasma cells, which are a highly differentiated state of B cells, synthesize and secrete antibodies.

T helper cells (Th) fulfill several important functions in the immune system. Th-cell-derived cytokines, as the onset of the immune signal, have been shown to determine which immune effector pathways are subsequently activated. Th cells are activated by reacting their antigen specific receptor with antigen presenting cells (APCs), having surface peptide fragments of treated foreign antigen bound to MCH class I I molecules. Thcell-activated cells secrete cytokines (lymphokines) that activate the respective immune effector mechanisms.

Th cells can be divided into three subgroups: ThO, Th1 and Th2, based on the type of cytokines secreted by them. (Fitch et al., Ann. Rev. Immunol., 11, pp. 29-48 (1993)). In mice, unstimulated naive T-helper cells produce IL-2. Th-stimulated cells are converted to ThO precursor cells, which produce a wide range of cytokines, including IFN-1, IL-2, IL-4, IL-5 and IL-10. Chronically stimulated ThO cells can differentiate to either Th1 or Tb2 cell types, thereby altering the type of secreted cytokine. Some cytokines, such as IL3, GM-CSF and TNF, will secrete both Th1 and T1p2 cells. Other cytokines are made exclusively from only one Th cell subgroup. (Romagnani etal., Ann. Rev. Immunol., 12, pp. 227-57 (1994)). Th1 cells produce LTa, IL-2, and IFN-γ, which activate macrophages and inflammatory responses related to cellular immunity and resistance to intracellular infections.

Th2 cells produce the cytokines IL-4, IL-5, IL-6 and IL-10, which enhance the cellular production of eosinophils and mast cells and help increase the number and maturation of B cells. (Howard et al., “T cell-derived cytokines and their receptors”, Fundamental Immunology, 3d ed., Raven Press, New York (1993)). Tb2 cells are also involved in the generation of B-cell memory, a somatic mutation, and thus affect affinity maturation and de novo regulation of isotype switching of immunoglobulin. For example, the Th2 cytokine IL-4 switches activated B cells to the synthesis of the IgG1 isotype and suppresses other isotypes. IL-4 also stimulates IgE overproduction in type I hypersensitivity reactions. Th2 cytokine IL-5 induces the synthesis of an IgA isotype important for mucosal immunity.

Secondary lymphoid tissues, such as lymph nodes (LN), spleen, and mucosal lymphoid tissues, are highly effective in capturing and concentrating foreign substances and are major sites for antigen-induced activation and differentiation of T and lymphocytes. These processes depend on the diversity and organization of the cells in these tissues, which in many respects provide a network of various manifestations of humoral immune responses such as T / B cell interactions, germinal center formation (GC), affinity maturation, switching immunoglobulin class and cell movement. (Klein, J., Immunology, John Wiley and Sons, (1982)). The molecular mechanisms responsible for the development, structural maintenance and function of peripheral lymphoid tissues have not been fully elucidated.

Although the underlying structure of secondary lymphoid tissues differs significantly and shows variation among mammalian species, the fine structure of these secondary lymphoid tissues has some common features, such as: (1) antigenic accessibility, (2) structural features that provide prolonged contact with lymphocyte antigen, (3) T-cell rich areas surrounded by B cells, (4) B cell rich follicles, (5) marginal zone type sites, (6) specialized endothelial cells and (7) sites for antibody production as discussed next passage below.

Secondary lymphoid tissues are available for antigen in the system. For example, the antigen reaches the spleen via sinusoidal blood flow, to the lymph node through the afferent lymphatic vessels, and is transported through a specialized epithelium into the mucosal lymphoid tissue.

Secondary lymphoid tissues in different organisms also have some common structural features, such as follicular dendritic cells (FDCs) and interdigital cells (IDCs), which ensure the long-term presence of the antigen in lymphocyte-rich tissue areas.

Another common feature is the presence of T-cell rich areas surrounded by B cells. For example, T-cell-rich regions include the peri-arteriolar lymphoid zones in the white spleen pulp and the paracortical area of the LN, which contain a large number of recirculating T cells and IDCs, which in turn function as auxiliary cells for T and B cells.

In addition, lymphoid tissues have typical B-cell-rich primary and secondary follicles in the white pulp of the spleen and in the cortex of the LN. The secondary follicles in these lymphoid tissues are also called germinal centers (GCs) and have a dense FDC network for trapping and presenting antigens.

Marginal area type areas have also been noted that define histological areas in mouse spleen and more diffuse sites in human secondary lymphoid organs. These areas mainly include marginal zone macrophages (MZM), metalophilic macrophages (MM), marginal zone B cells and reticular cells, but may also include T cells and dendritic cells. (Kraal, Int. Rev. Cytol., 132, pp. 31-74, (1992)). Opening of the arterial blood flow in the marginal area leads to direct access of antigens to these cells and induces cellular responses to the antigen at this site. (Kraal, Int. Rev. Cytol., 132, pp. 31-74, (1992)). The presence of MZM is also necessary for the optimal movement of B cells in the white pulp of the spleen. (Kraal, 1992; Kraal, etal., Immunology, 68, pp. 227-232, (1989)).

As a rule, blood flow lymphocytes enter the secondary lymphoid tissues, passing through a special endothelium, such as the LN (high endothelial venous - HEV) endothelium endothelium and the endothelium that undergo splenic blood sinus in similar structures to the margins. This endothelium expresses adhesive molecules and addresses that function in the movement of cells to secondary lymphoid tissues. For example, peripheral LN addresses (PNAd) are different from the mucosal LN addressin, MAdCAM-1, which is involved in the movement of lymphocytes to mucosal lymphoid tissues, including tissues such as mesenteric LN, Peyer's plaques and lamina propria.

Not all addresses are clearly defined, for example, the adrenal gland lymphocyte adrenal gland is not defined. The physiological role of these addressees involves an increased requirement for an appropriate group of antigen-specific lymphocytes to be involved in the immune response and the subsequent spread of the immune response throughout the body.

Finally, plasma cells, which are plasma cells that produce antibodies, are detected at different sites in which the Antigen precursor cells are activated. For example, antibody-producing plasma cells in the red spleen of the spleen are derived primarily from B-cell activation from the T cell zones, and plasma cells in the LN medulla are derived from B cells activated in the T-cell zones of the same lymph node. Similarly, antibody-producing plasma cells in bone marrow are derived from B cells activated in the spleen and lymph node, and plasma cells in the lamina propria of the intestinal tract originate mainly from B cells activated in mesenteric LN or lymphoid tissue of the intestine.

See, e.g. ICM MacLennan, "The Structure and Function of Secondary Lymphoid Tissues" in Clinical Aspects of Immunology, 5 ^th edition, eds. PJ Lachman, Sir DK Peters, FS Rosen, MJ Walport, Blackwell Scientific Publications, pp. 13-30, (1993).

In general, the cellular / histological events underlying the humoral immune response to T-dependent antigens are as follows (Toellner, et al., J. Exp. Med., 183, pp. 2303-2312, (1996)):

In the inductive phase, naive B and T cells activate and engage in the immune response several days after the antigen enters the body. For example, within the spleen within 12 hours of secondary response immunization, the memory cells meet with the blood antigen in the marginal zone and leave the marginal zone to go to the T cell zones. B cells can be detected in the T cell zones within 24 hours. Immunoglobulin transcripts can be detected within 12 hours of re-exposure to the antigen, which means that T-B cell interaction has already taken place. The B cells then migrate to the outlet regions and the red pulp where they proliferate and form centers of B cell blasts and differentiate into plasma cells. B cells also continue to proliferate in the IDC-rich T-cell region. Within 4 days after immunization and after GC proliferation, production of memory B cells begins. In the initial response, well-developed GCs are seen at 10 days and reach a maximum at 14 days after immunization.

T-cell proliferation in T-cell zones is observed at 48-72 hours and peaks at 7 days after immunization. This T cell proliferation promotes T cell-dependent B cell activation. The proliferative levels in the T-cell zone decrease as GCs are formed. T-cell proliferation is also found in GC, where centrocytes (B cells) from the dark zone capture the antigen from I DC and present it to the T cells in the light zone.

The T cell-dependent antigen can activate B cells from the marginal zone, newly acquired naive B cells, and recirculating lymphocytes, attracted to and retained in secondary lymphoid organs, by addressing and adhesive molecules. Naive B cells show the same kinetics for movement to the T cell zone, ie, which is also characteristic of activated B cells.

Phase of T-Cell Dependent Responses established

The established phase of T-cell-dependent responses is supported by the prolonged activation of memory B cells in the secondary lymphoid follicles. At this stage, there is very little increase in the number of naive B cells and the response is mainly due to an antigen retained by the FDC. GCs are required for optimal memory generation, isotype switching, somatic mutation, and therefore affinity maturation of immunoglobulins.

Increasing such lymphocyte responses results in antibodies capable of circulating in the body in different pathways, for example, antibodies leave the spleen through the bloodstream and leave the LN via efferent lymphatic pathways. Thus, antibodies meet and bind directly to the invading pathogen. This recognition process unlocks a cascade of immune effector mechanisms involving activation of the complement cascade and cellular responses mediating the host's protection against the pathogen.

Antibodies also play a role in some pathological responses, such as hypersensitivity responses - inappropriate or disproportionate immune responses elicited by contact with a previously encountered antigen. There are four distinctive types of hypersensitivity.

Type I "immediate type hypersensitivity" involves the activation of an allergen-induced TK2 cell and the release of a Th 2 cytokine. Th2 cytokine IL-4 stimulates 3 cells to undergo isotype switching to produce IgE, which in turn activates mast cells, which cause acute inflammatory responses such as those leading to eczema, asthma and rhinitis.

Type II and III hypersensitivity is caused by IgG and IgM antibodies directed against cell surface antigens or specific tissue antigens (Type II), or soluble serum antigens, with which they form circulating immune complexes (Type III).

Type IV "delayed type" hypersensitivity (DTH) is a Th1 cell-mediated response and can be transmitted between mice by Thl cell transfer but not by serum transfer alone. This characteristic distinguishes Type IV DTH, unlike the other three types of hypersensitivity, which require humoral immune responses, and is caused mainly by antibodies that can be transmitted in serum-deprived cells. (Roitt et al., Immunology, pp. 19.122.12 (Mosby-Year Book Europe Ltd., 3d ed., 1993)).

Pathological humoral immune responses are associated with organ-specific and systemic autoimmune conditions, such as Systemic Lupus Erythematosus, Wegener's Granulomatosis, Polyarteritis Nodosa (PAN), Rapidly Progressive Crescentic 'Glomerulonephritis, and Idiopathic Thrombocytopenia, as well as disease, Chagas' disease. "Humorous immune responses can also contribute to rejection of a grafted tissue or organ transplant.

So far immunomodulators and immunosuppressive agents have been used to treat these various immunological cases. The three main immunosuppressive agents currently used are steroids, cyclophosphamide and azathioprine.

Steroids are pleiotropic anti-inflammatory agents that suppress activated macrophages and inhibit the activity of antigen presenting cells in ways that cancel out many pathological T cell effects. Cyclophosphamide, an alkylating agent, mediates cell death by repressing DNA replication and repair. Azathioprine is an anti-proliferative agent that inhibits DNA synthesis. These nonspecific immunosuppressive agents are usually required at high doses, which increases their toxicity (ie, nephro- and hepatotoxicity) and has deleterious side effects. Therefore, they are not suitable for long-term therapy.

This means that additional agents and therapies are needed to overcome the problems caused by conventional treatment.

SUMMARY OF THE INVENTION

The present invention solves the above problems by delivering pharmaceutical compositions and methods for treating immunological diseases by suppressing lymphotoxin-B receptor (LT-B-R) signaling using lymphotoxin B receptor blocking agents. In particular, compositions and methods comprising LT-BR blocking agents are useful for inhibiting antibody-mediated immune responses to regulate the levels of expression of cellular adrenals and cell movement, to influence the differentiation of follicular dendritic cells, and to alter the structural organization of secondary and secondary structures tissues and similar lymphoid structures occurring in pathological cases, such as systemic lupus erythematosis and idiopathic thrombocytopenia purpura. In addition, in some embodiments of the invention, it is necessary to alter the association between immune complexes and B cells. In particular, the methods of the invention may prevent the presentation or deposition of antigens on cells, or alternatively, completely dissolve or delete antigens already present on cells.

Alternatively, the LT-B-R blocking agent is selected from the group consisting of soluble lymphotoxin-B-R, an antibody directed against LT-B-R, and an antibody directed against a surface LT ligand.

In one embodiment, soluble forms of the lymphotoxin-β receptor extracellular domain are proposed that act as LT-B-R blocking agents. Preferred compositions and methods of this embodiment include a recombinant lymphotoxin-β receptor fusion protein having the Ι-Τ-β-R extracellular ligand binding domain coupled to the heavy chain constant portion of the immunoglobulin domain. Preferably, the Ι-Τ-β-R ligand binding domain is fused to the human IgG Fc domain.

In another embodiment of the present invention there are provided antibodies that act as Ι---β-R blocking agents. Preferred compositions and methods of this embodiment include one or more antibodies directed against the lymphotoxin-β receptor. Preferably, the antibody is monoclonal. Other preferred compositions and methods of this embodiment include one or more antibodies directed against surface lymphotoxin. Preferably, the antibody is a monoclonal antibody directed against lymphotoxin-β. Preferred antibodies include anti-human ΒΤ-β-R mAb BDA8 and anti-human LT-β mAb B9.

In other embodiments, the invention relates to methods of altering the humoral immune response in an animal by administering a pharmaceutical composition having a therapeutically effective amount of an LT-B-R blocking agent. In some other embodiments, the pharmaceutical composition is administered in an amount sufficient to cover LT-B-R positive cells for about 1 to 14 days. The pharmaceutical composition may optionally include, further, a pharmaceutically acceptable carrier or adjuvant.

In other embodiments, methods are proposed to suppress LT-B-R signaling without inhibiting TNF-R signaling using the Ι-Τ-β-R blocking agents described above. The invention also encompasses methods for treating, preventing or eliminating human immunodeficiency virus in mammals, including the administration of blocking agents by LT-BR or alone or together with pharmaceutical carriers, adjuvants, or other preparations known to those skilled in the art, as useful in treating or ameliorating the symptoms of HIV or spin.

In addition, the present invention relates to methods of treatment in the field of transplantation, i. graft rejection. In particular, some embodiments relate to the simultaneous introduction of a blocking agent from the CD40 pathway and a blocking agent from the LT pathway.

BRIEF DESCRIPTION OF THE GRAPHS

Figure 1 is a sequence of the extracellular portion of the human LT-B receptor that encodes a ligand binding domain.

Figure 2 is an immunohistochemical analysis of the spleen of mice that have been repeatedly injected with LTB-R-Ig or LFA-3-Ig hybrid proteins and antigen.

Figure 3 is an immunohistochemical analysis showing the absence of germinal centers of spleens of LTB-R-Ig treated and MR1 (antiCD40 ligand antibody) treated mice and the presence of follicular dendritic cells in sprays of MR-1 but not of LTB-R- lg treated mice. Hybrid proteins and SRBC (sheep erythrocyte antigen) antigen were introduced as described in Figure 2.

Figure 4 is an immunohistochemical analysis showing that the expression of addressin changes in the LN of mice treated in utero and long after birth with LTB-R-Ig.

Figure 5 is an immunohistochemical assay for lymphocyte placement and macrophage marker expression, in mesenteric LN of mice treated (as in Figure 4) in utero and long after birth with LTB-R-Ig.

Figure 6 is an immunohistochemical analysis showing that treatment of mice with LTB-R-Ig suppresses the antibody response to SRBC.

Figure 7 represents an immune complex captured by FDCs.

DETAILED DESCRIPTION OF THE INVENTION

In order for the invention described herein to be fully understood, the following detailed description is set forth.

The term "immunoglobulin response" or "humoral response" as used herein refers to an animal's immunological response to a foreign antigen by which the animal produces antibodies to the foreign antigen. The Th2 class of T helper cells is important for the efficient production of high affinity antibodies.

The term "germinal center" as used herein refers to a secondary B-cell follicle that is formed after antigen immunization. The appearance of this histologic area correlates with the construction of optimal memory, isotype switching, somatic hypermutation, sequential affinity maturation of the antibody response.

The term "marginal zone" or "type region - marginal zone" refers to a histologically characteristic area of secondary lymphoid tissues, including primarily macrophages from the marginal zone (MZM), metapophilic macrophages (MM), B cells from the marginal zone and the reticular cells as well as T cells and dendritic cells. The arterial blood flow opens into the marginal sinuses, thus delivering the antigens directly to these cells and facilitating cellular responses to the antigens in the area.

The term "addressin" as used herein refers to a molecule involved in the movement of lymphocytes to secondary lymphoid organs. Such molecules are expressed on endothelial cells, especially in high endothelial venules in the lymph nodes. The spleen's address is not defined. MAdCAM-Ι is mucosal addressin; PNAd is a peripheral addressin.

The term T-helper (Th) cells, as used herein, refers to a functional subclass of T cells that help to form cytotoxic T cells and which cooperate with B cells to stimulate antibody production. Helper T cells recognize the antigen bound to class II MHC molecules and promote contact-dependent and independent (cytokine) signals to effector cells.

The term "cytokine" as used herein refers to a molecule that mediates signaling between cells. "Lymphokine" is a cytokine secreted by lymphocytes.

The term "Th2" refers to a subclass of T helper cells that produce LTa, interferon-γ, and IL-2 (and other cytokines) and that trigger cell-related inflammatory responses, i. non-immunoglobulin response to a signal.

The term "Th2" refers to a subclass of T helper cells that produce cytokines, such as IL-4, IL-5, IL-6, and IL-10, which are linked to the immunoglobulin (humoral) response to an immune signal.

The term "Fc domain" of an antibody refers to a portion of the molecule comprising the hinge region, the CH2 and CH3 domains, but which lacks antigen binding regions. It is also understood that the term includes equivalent regions of IgM or other antibody isotypes.

The term "anti-LTB receptor antibody" refers to any antibody that specifically binds to at least one epitope of the LTB receptor.

The term " anti-LT antibody " refers to any antibody that specifically binds to at least one epitope of LTa, LTB, or the LTo / B complex.

The term "LTB-R signaling" refers to the molecular reactions associated with the LTB-R pathway and the subsequent molecular reactions that follow.

The term "LTB-R blocking agent" refers to an agent that can attenuate ligand binding to LTB-R, to groups of cell surface LTB-R or LTB-R signaling, or which may interfere with the interpretation of the LTB-R signal in the cell.

The LTB-R blocking agent, which acts at the stage of binding of the ligand to the receptor, can suppress the binding of the LT ligand to LTB-R by at least 20%. Examples of LTB-R blocking agents include, soluble LTB-R-Fc molecules and anti-LTa, anti-LTB, antiLT cx / β and anti-LTB-R Abs. Preferably, the antibodies do not cross-react with the secreted form of LTa.

The term "LTB-R biologically active" refers to: 1) the ability of an LTB-R molecule or derivative to compete for a soluble or surface LT binding ligand with soluble or surface LTB-R molecules; or 2) to native LTB activity, such as one that is capable of stimulating an immune regulatory response or cytotoxic activity.

The term "LT ligand" refers to the LTa / β heterodimer complex or derivative thereof that can specifically bind to the LTB receptor.

The term '' LTB-R ligand binding domain '' refers to a portion or parts of LTB-R that are involved in the specific recognition and interaction with the LT ligand.

The term "surface LT" and "surface LT complex" refers to a complex comprising LTa and membrane-bound LTB subunits - - ^ including mutant, altered and chimeric forms of one or more of the subunits - that is located on the cell surface. &Quot; Surface LT ligand " refers to a surface LT complex or derivative thereof that can bind specifically to the LTB receptor.

The term "object" refers to an animal or to one or more cells derived from an animal. The animal is predominantly a mammal. Cells may be in any form, including, but not limited to, cells retained in tissue, groups of cells, immortalized, transfected or transformed cells and cells derived from an animal that has been physically or phenotypically altered.

Lymphotoxin β; Member of the TNF Family

Cytokines similar to Tumor Necrosis Factor (TNF) have emerged as a large family of pleiotropic mediators in host defense and immune regulation. Representatives of this family exist as membrane-bound forms that act locally through cell-cell contact or as secreted proteins that can act on distant targets. In parallel, a family of TNF-like receptors interact with these cytokines and unlock different pathways including cell death, cell proliferation, tissue differentiation, and inflammatory responses.

TNF, lymphotoxin a (LTcx, also called TNFB) and lymphotoxin β (LTB) are members of the TNF ligand family, which also include ligands to the Fas, CD27, CD30, CD40, OX-40 and 4-1BB receptors. (Smith et al., Cell, 76, pp. 959-62, (1994)). Signaling by several TNF - - ^ family members, including TNF, LTa, Ι_Τβ, and Fas - -, can cause tumor cell death by necrosis or apoptosis (programmed cell death). In non-tumorigenic cells, TNF and many of the TNF family of ligand-receptor interactions influence the development of the immune system and the responses to various immune signals.

Most membrane-bound LTa / β complexes (“surface LT”) have Ι_Τα1 / β2 stoichiometry. (Browning et al., Cell, 72, pp. 847-56, (1993); Browning et al., J. Immunol., 154, pp. 33-46, (1995)). Surface LT ligands do not bind with high affinity to TNFR and do not activate TNF-R signaling. However, the LTB receptor (LTBR) binds to these high affinity surface lymphotoxin complexes (Crowe et al., Science, 264, pp. 707-10, (1994)).

LTB-R signaling, similar to TNF-R signaling, has antinchroliferative effects and can be cytotoxic to tumor cells. Compositions and methods for selectively stimulating LTB-R using LTB-R activating agents are reported in the accompanying U S application Serial Number 08 / 378,968. LTB-R activating agents are used to suppress tumor cell growth without co-activating TNFR-induced pro-inflammatory or immunoregulatory pathways.

New gene studies suggest the role of LTα / β in the development of secondary lymphoid organs. (Banks et al., J. Immunol., 155, pp. 1685-1693, (1995); De Togni et al., Science, 264, pp. 703-706, (1994)). Indeed, LTot-deficient mice do not have lymph nodes (LN) and Peyer's plaques (PP). In addition, their spleens have a broken structure and the expression of functional markers on cells from the marginal area of the spleen is altered. (Banks et al., 1995; De Togni et al., Science, 264, pp. 703-706, (1994); Matsumoto et al., Science, 271, pp. 1289-1291, (1996). None of these characteristics has been described for the TNF receptor and knockout mice. (Erickson et al., Nature, 372, pp. 560-563, (1994); Pfeffer et al., Cell, 73, pp. 457-467, (1993); Rothe et al., Nature, 364, pp. 798-802 (1993). Recently, applicants have identified the role of membrane LTcx / β complexes in the development of secondary lymphoid organs, showing that in the generation of mice that were injected during pregnancy with the soluble form of the murine LTB-R bound to the Fc portion of human lgG1 (LTBR-Ig), most lymph nodes are missing and the structure of the spleen is damaged. (Rennert et al., 1996, “Surface Lymphotoxin alpha / beta complex is required for the development of peripheral lymphoid organs. J. Exp. Med. 184: 1999-2006). In another study of transgenic mice for a similar LTB-R-Ig construct, which begins to be expressed three days after birth, they are shown to have LN. However, the structure of their spleen is damaged and several markers are not expressed in the cells from the marginal area of the spleen. (Ettinger et al., “Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble LTB-R / lgG1 fusion protein”. Proc. Natl. Acad. Sci., U S, 93: 13102-7). These data indicate that it takes time for the membrane functions of LT to mediate effects directed at the development of secondary lymphoid organs but not effects on the structure of the spleen.

The TNF system may also function in splenic development. Cells from the marginal area of the spleen in TNF-deficient mice do not express macrophage markers or MAdCAM-

1. (Alexopoulou et al., 60 ^{th Int. TNF Congress, Eur.} Cytokine Network, pp. 228 (1996); Pasparakis et al., 60 ^{th Int. TNF Congress, Eur.} Cytokine Network, pp. 239, ( 1996). TNF-R55 deficient mice also lack MAdCAM-1 (but not MOMA-1), found when staining the marginal area of the spleen (Neumann et al., J. Exp. Med., 184, pp. 259-6). 264, (1996), Matsumoto et al., Science, 271, pp. 1289-1291, (1996). As seen in the spleen of TMP-P75-deficient mice, expression of these markers appears to be normal. (Matsumoto et al. ., Science, 271, pp. 1289-1291, (1996).

Lymphoid-like tissues not only occur as part of developmental processes, but also appear in some pathological cases, such as chronic inflammation, a process ₇ recently called neolympho-organogenesis. (Picker and Butcher, Annu. Rev. Immunol., 10, pp. 561-591, (1992); Kratz, et al., J. Exp. Med., 183, pp. 1461-1471, (1996)). Apparently, these processes are influenced by representatives of the TNF family. Transgenic mice with respect to the LTa gene, driven by the rat insulin promoter (RIP-LT), develop LT-induced chronic inflammatory lesions, with characteristics of organized lymphoid tissues. (Kratz, et al., J. Exp. Med., 1183, pp. 1461-1471, (1996); Picarella et al., Proc. Natl. Acad. Sci., 89, pp. 10036-10040, (1992 ).

Assessment of LT function during the T cell-dependent immune response, using LTa-deficient mice, indicates the need for LT to form GC, possibly to maintain organized follicular dendritic cell structure (FDCs) and for humoral responses. (Banks et al., J. Immunol., 155, pp. 1685-1693, (1995); Matsumoto et al., Science, 271, pp. 1289-1291, (1996); Matsumoto et al., Nature, 382 , pp. 462-466 (1996). TNFR55-deficient mice also lack FDCs, do not develop GC, and do not develop optimal antibody response to sheep erythrocyte cells (SRBC). This suggests that TNF-R55 can be unlocked by soluble LT or TNF signals for most of these responses. (Le Hir et al., J. Exp. Med., 183, pp. 2367-2372, (1996); Alexopoulou et al., 60 ^th Int. TNF Congress, Eur. Cytokine Network, pp. 228, (1996) Pasparakis et al., 60 ^th Int. TNF Congress, Eur. Cytokine Network, pp. 239, (1996)). To date, the functional role of the surface LT / LTBR pathway in humoral immune responses has not yet been defined.

The LTB receptor, a member of the TNF receptor family, binds specifically to surface LT ligands. LTB-R binds to heterodimeric LT complexes (mainly LTa1 / B2 and LTo: 2 / B1) but not TNF or LTa. (Crowe et al., Science, 264, pp. 707-10, (1994)). LTB-R mRNAs are detected in human spleen, thymus and major organs involved in the immune system. Although LTB-R expression studies are in their infancy, LTB-R expression patterns appear to be similar to those known for TNFR55, except that LTB-R is absent on peripheral T and B blood cells and T and B cell lines.

Cell surface lymphotoxin (LT) complexes are characterized in CD4 + T cell hybridoma cells (11-23. D7), which express high levels of LT. (Browning et al., J Immunol., 147, pp. 1230-37, (1991); Androlewicz et al., J. Biol. Chem., 267, pp. 2542-47, (1992), two of which are included here). The expression and biological role of LTB-R, LT subunits, and surface LT complexes are reviewed by C. F. Ware et al., “The ligands and receptors of the lymphotoxin system,” in Pathways for Cytolysis, Current Topics Microbiol. Immunol., Springer-Verlag, pp. 175-218, (1995) specifically incorporated herein by reference.

LTa expression is induced and LTa is secreted mainly by activated T and B lymphocytes and natural killer cells (NK). Among T helper cells, LTa appears to be produced by Th1 but not by Th2 cells. Also, LTa is detected in melanocytes, microglia and T cells in patients with multiple sclerosis lesions that respond with anti-LTa antisera (Selmaj et al., J. Clin. Invest., 87, pp. 949-954 , (1991).

Lymphotoxin B, (also called p3Z) is expressed on the surface of human and murine T lymphocytes, T cell lines, cell lines and lymphokine-activated killer (LAK) cells. LTB is the subject of applicants for accompanying international pending applications PCT / US91 / 04588, published January 9, 1992, as WO 92/00329; and PCT / US93 / 11669, published June 23, 1994, as WO 94/13808, which are incorporated herein by reference.

Superficial LT complexes are mainly expressed by activated T (helper, Th1 and killer cells) and B-lymphocytes and natural killer cells (NK), which is determined by FACS analysis or immunohistologically using anti-LTa antibodies or soluble LTB -R-Ig hybrid proteins. Compositions and methods for the use of soluble LTB receptors and anti-LTB receptor and ligand-specific antibodies as therapists for Th1 mediated immunological diseases are disclosed in the accompanying pending US application Serial Number 08 / 505,606, filed July 21, 1995. cells. Superficial LT has also been described on human cytotoxic T-lymphocyte (CTL) clones, activated peripheral, mononuclear lymphocytes (PML), 1L-2 - activated peripheral blood lymphocytes (LAK cells), pokeweed mitogen-activated or anti-C040 -Lymphocytes (PBL) and various T and B-cell lymphoid tumors. The incorporation of target alloantigen-bearing cells induces specific expression of LT by CD8 + and CD4 + CTL clones.

Applicants have described here several immunological functions for surface LT and have shown the effects of LTo / β binding reagents on the origin and nature of immunological responses, maintaining the cellular organization of secondary lymphoid tissues, including effects on the differentiated state of follicular dendritic cells and dendritic cells centers and expression levels of adrenaline that affect cellular movement. Applicants thus determine the therapeutic applications of surface LTcs / β and LTB receptor binding agents.

Until the present invention, however, the effect of LTB-R signaling on humoral or immunogenetic responses has not been fully understood. For the first time, the invention discloses that if the LT pathway is blocked by LT-B or LTB-R, the humoral immune response can be altered in the animal. Accordingly, in a broader embodiment, the invention relates to methods of altering the humoral immune response in an animal, comprising the steps of administering a pharmaceutical composition that comprises an effective therapeutic amount of a LT pathway blocking agent, preferably LTB-R blockers.

Any blocking agent can be used in the invention, but those skilled in the art can very easily identify agents that block LTB-R. For example, such blocking agents may include small molecules, receptor inhibitors, soluble lymphotoxin-B receptor, antibodies directed against LTB-R, and antibodies directed against the surface LT ligand. In preferred embodiments, blocking agents include soluble LTB-R1 having a ligand binding domain that can selectively bind to a surface LT ligand, most preferably one wherein the soluble LTB-R includes a human immunoglobulin FC domain.

In other embodiments, preferred blocking agents include monoclonal antibodies directed against LT-B-R, comprising predominantly anti-human LTB-R mAb BDA8 and anti-human LT-B-mAb B9. More preferred antibodies include, A1.D5.18 and AO.D12.10 and several examples of BB-F6ln. it is desirable to use a monoclonal antibody directed against a murine surface LT ligand.

Prolonged presentation of an antigen by FDCs is probably important in autoimmune diseases, where prolonged activation of the immune system by endogens or autoantigens leads to disease continuation. The immune complex captured by FDC's is illustrated in Figure 7. The ability to remove these immune complexes from FDC will result in a decrease in immune activity and suppression of the disease or even a halt in the development of the disease. Those autoimmune diseases that include abnormal antibody responses are clearly targeted for LT pathway inhibitors, although other more classically T-cell-mediated autoimmune diseases may have unrecognized humoral components and may therefore also have beneficial effects .

Likewise in the field of transplantation, graft rejection, i. host versus transplant disease and host transplant disease requires constant antigen presentation. The FDC manipulation mechanisms described herein can also be applied to problems related to the recognition of foreign ones, i. of transplants.

In addition, prolonged presentation of the antigen or maintenance of antigen memory may be relevant for these autoimmune diseases caused by molecular mimicry. For example, the immune response to the Lyme disease infectious agent Borrelia burgdorferi, leads to arthritis - a similar disease, most likely because some antigenic epitope on this bacterium resembles a normal joint component. Removal of the FDC-retained Lyme bacterial antigen may alleviate Lyme disease-induced arthritis. This therapy may also be appropriate in other cases of mimicry associated with infectious agents.

Most unexpectedly, the Applicants have found that the administration of blocking agents for LTB-R may interfere with the presentation and / or deposition of antigens on follicular dendritic cells. Generally, B cells recognize the antigen as immune complexes bound to the surface of follicular dendritic cells. Follicular dendritic cells can retain the antigen indefinitely. Thus, periodic contact with the antigen retained on the FDC may be associated with the retention of B-cell memory. Therefore, the methods of the claims cover a large number of disease states that are dependent on the presentation of an antigen on dendritic cells. The introduction of blocking agents of the invention may occur prior to the introduction of an antigen into the animal, in which case blocking agents will prevent the deposition of all or part of the antigen on the follicular dendritic cells, thereby inhibiting or attenuating the expected immunogenic response. Accordingly, the blocking agents of the invention may be introduced into the animal at some point after the follicular dendritic cells have bound to the antigen. With the methods claimed, this linkage can be broken, so that the expected immunogenic response can then weaken or disappear. Therefore, the therapeutic methods of the invention may include the elimination of all or part of the immune complexes already trapped in the B cell follicles, or the protection of whole or part of the captured immune complexes on the B cell follicles.

The ability to disrupt the association between these antigen presenting follicular dendritic cells and immune complexes is probably unique to the LT-β pathway. For example, anti-CD04_ (MR-1) is another member of the TNF family and is also expressed on follicular dendritic cells. Like LT-B-R / Ig, MR-1 has been shown to prevent germ cell formation but does not affect the expression of FDC markers. Ahth-CD40-L, unlike LTB-R, does not interfere with the capture of immune complex on follicular dendritic cells, nor is it capable of eliminating immune complexes pre-captured on follicular dendritic cells. In addition, the applicants show that anti25

CD40-L does not affect the survival / maintenance of preformed memory B cells.

Although the exact basis is unknown, for the differences between the effects of anti-CD0-1-1 and LT-B-R blocking agents, it is hypothesized that CD40 may deliver cell survival signals. However, the LT system is critical for the maintenance of follicular dendritic cells in a fully differentiated and functional state, a condition that seems necessary for germinal center response, for the formation and maintenance of B cell memory. Therefore, blocking the CD40 / CD40L pathway may prevent the formation of memory B cells, but will not affect the memory pool already established. On the other hand, blocking the LT pathway not only impedes the formation and maintenance of memory B cells, but also affects the maintenance of previously formed memory B cells.

Another application to the LT pathway suppression lies in the treatment of viruses that form reservoirs in the follicular dendritic cell (FDC) compartment. HIV is a good example of this. As a result of the viral infection, large amounts of the infectious virus remain on FDC's, in the extracellular follicles of the secondary lymphoid organs. (Heathe et al., 1995, "Follicular dendritic cells and human immunodeficiency virus infectivity.", Nature, 377: 740-4). The virus is thought to be in complex or complement or immunoglobulin and is bound to either Fc or complement receptors or both. Therefore, the virus uses the normal mechanism of the immune system to overcome antigenic memory for long periods. During the disease, mainly in these places, active lymphocyte infection occurs. During the asymptomatic phase of infection, the pool of the virus in this compartment was estimated to be more than 10 times larger than that contained in T cells and monocytes. (Carvert et al., 1997, "Kinetics of response in lymphoid tissues to antiretroviral therapy for HIV-1 infection", Science, 276: 960-4). In modern treatments for HIV, many antiviral agents are combined to reduce the amount of the virus and to avoid the emergence of resistant variants. A possible limitation of this therapy is its imperfection, because during these intervals, the remaining virus can mutate, allowing the development of resistant variants and thus disrupting the therapeutic process. When the amount of virus in the FDC compartment is dramatically affected by repeated drug therapy, the drugs themselves act primarily on the viral replicating machine rather than the non-replicating virus on the surface of the FDC. Therefore, the viral reservoir on FDC's may serve as a re-inoculum after discontinuation of drug therapy. In addition, FDCs can transform the neutralized virus into an infectious form, further emphasizing the importance of these cells in the pathogenesis of HIV.

Because inhibition of the LT pathway can result in the release of FDCs from immune complexes from the cell surface, it can also release HIV in the form of an immune complex. It is desirable to release the entire amount of HIV in this compartment immediately before the start of the designated multiple therapy, with the released virus either to be processed and removed from the body, or the infectious virus to be susceptible to drug therapy. This combination can reduce the remaining amount of the virus to very low levels, which probably helps the treatment. In this case, either LTBR / Ig or blocking antibodies to the ligand or receptor will be beneficial. A possible treatment protocol would initially include drug therapy and then, within a few days, the release of each associated virus by one or more inhibitors of LT pathway inhibitors. Once the amount of the virus has already been reduced, further treatment with an anti-LT agent is not required.

While HIV is a particularly well-researched example, other viruses may reside or lurk on FDC's in a latent state, expecting some event as an immunological disorder that results in large amounts of additional antigenic burden and subsequently release of the associated virus from FDCs and new appearance of the virus. Therefore, this invention relates to all LT pathway inhibitors in order to avoid complications of FDC-related virus.

This finding has significant implications for a large number of diseases due to the presentation of antigen on dendritic cells and the response produced by memory B cells. Effective LTa1 / B2 signaling serves as an example of therapeutically useful anti-LTb blocking monoclonal antibodies. In addition, an anti-human LT alpha monoclonal antibody, called AOD12, can conveniently block 1_TcI / B2 signaling, but unlike most anti-human LT alpha monoclonal antibodies, it is poorly effective against LTa only. These monoclonal antibodies were obtained after immunization of mice with soluble LTa1 / B2 ligand, which led to the detection of monoclonal antibodies with unique specificity. In addition, we argue that anti-LTcx monoclonal antibodies with specificity directed primarily against the 1_Ta1 / B2 complex will only be detected in this immunization mode and not by LT alpha immunization alone and, therefore, it includes a unique class of anti -La antibodies.

EXAMPLES FOR THE IMPLEMENTATION OF THE INVENTION

Materials and methods

Mice

Pregnant Balb / c mice were supplied by Jackson Laboratory (Bar Harbor, ME), enclosed, and treated according to the instructions in the instructions. Receptor-Ig proteins or mAbs are injected into the tail vein (iv) of pregnant mice. A generation of these mice and 5-week-old female Balb / c mice (delivered by Jackson Laboratory, Bar Harbor, ME) were injected with hybrid proteins intraperitoneally (ip).

Hybrid Proteins and Antibodies

Hybrid proteins are prepared comprising the extracellular domain or from murine LTB-R human TNF-R55 or human LFA-3 (which does not bind to murine CD2) hinged to the C _n 2 and C _n 3 domains of human lgG1 as described from (Force et al., J. Immunol. 155, pp. 5280-5288, (1995); Miller et al., J. Exp. Med., 178, pp. 211-222, (1993)). Purified human IgG1 used as a control was supplied by Protos Immunoresearch (San Francisco, CA). MR1, an anti-mouse CD40 ligand antibody, was supplied by Pharmingen (San Diego, CA).

Antibodies (MOMA-1, ED3) specific for markers expressed by murine metallophilic macrophages (MM), (ED3 recognizes sialoadhesin) or murine specific reticular fibroblasts (ER-TR-7) were supplied by Serotec (Oxon, UK) . Mouse specific B220, CD4 and MadCAM-1 antibodies (MECA 367) were supplied by Pharmingen (San Diego, CA). An antibody (EP-TP-9) specific for a marker expressed by murine macrophages from the marginal zone was provided by Dr. Reina Mebius (Vrije Universiteit, Amsterdam). Antibodies (FDC-M1 and FDC-M2) specific for mouse follicular dendritic cell (FDC) have been described previously (Maeda et al., J. Immunol., 148, pp. 2340-2347, (1992)). An antimicrobial CR1 antibody (which also stains FDC) was kindly provided by Dr. Randolph J. Noelle (Dartmouth Medical Scool). MESA 79 antibody (cell culture supernatant obtained from cells supplied by ATCC, Rockville, MD) was used to detect peripheral lymph node (PNAd) addressing.

Antigens and Immunizations

Mice were immunized with 100 μΙ 10% suspension, ip, by SRBC (supplied by Colorado Serum Company). This is equivalent to 1 -5x10 ⁸ SRBC for immunization.

And his nohistochemistry

Lymph nodes and spleen freeze in OCT including medium (Miles, Elkhart, ΙΝ) _? π are mounted ^ to make cryostat sections. Sections 7-10 mm thick are dried and fixed with acetone. The sections were then incubated with conjugated antibodies for 1 hour at room temperature in a humidified vessel after dilution with Tris salt buffer A (TBS-A, 0.05M Tris, 0.15M NaCl, 0.05% Tween-20 (v / v), 0.25 % bovine serum albumin (BSA)), washed in TBS-B (0.05M Tris, 0.15M NaCI, 0.05% Tween-20) and fixed for 1 minute in methanol before the enzyme reaction began. Peroxidase (horseradish HRP) activity and that of alkaline phosphatase (AP) were manifested using a DAB tablet substrate kit (Sigma, St. Louis, MO) and 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT, Sigma) respectively. Tissue sections were fixed for 5 minutes in methanol and stained with Gimza (Fluka, Buchs, Switzerland).

Fluorescence Image Analysis

For immunofluorescence staining, frozen sections were fixed in acetone, air-dried and pre-blocked with 5 µg / ml 1HTH-CD16 / CD32 Fc blocker (Pharmingen, San Diego CA) in a buffer buffered with 0.25% BSA, 0.05% Tween-20 and 10% heated aggregate rabbit serum. Sections were stained in the same buffer using the following monoclonal antibodies (mAbs) and developing solutions: 10 .ug / ml biotinylated anti-B220 gPAl (Pharmingen), followed by 20 ug / ml streptavidin-FITC (Southern Biotechnology Associates, Birmingham, AL); 10 .ug / ml from MESA 367, followed by 10 µg / ml PE-goat F (ab ') 2 anti-rabbit IgG (Southern Biotechnology Associates); culture supernatant of MESA 79 followed by 20 µg / t! FITC mouse anti-rabbit IgM (Pharmingen); 20 µg / ml anti-sialoadhesin mAb followed by 10 µg / ml PE-goat F (ab ') 2 anti-rabbit IgG (Southern Biotechnology Associates), 50 µg / ml biotinylated PNA (Vector Laboratories, Burlingame, CA). followed by 10 µg / ml streptavidin-PE (Southern Biotechnology Associates); 1: 5 diluted mAb MOMA-1 with cell supernatant followed by 20 μg / ml FITC-mouse anti-rabbit IgM (Pharmingen). Some sections are stained with many mAbs simultaneously, allowing detailed image analysis. All sections were visualized at 50x optics and photographed using Ektachrome P1600 (Kodak, Rochester, NY) or obtained as an image of separate red or green files, as described by (Rennert et al., J. Exp. Med (November 1996, in press).

Hemagglutinin analysis

Serum dilutions were made in 96 well microtiter plates (Costar, Cambridge, MA) in PBS / 1% glucose.

The SRBC-specific IgM titer was determined by adding a 25 μΙ suspension of 10% SRBC in each well and the plate incubated for 1 hour in a 37 ° C incubator. For SRBC-specific IgG, serum was incubated for 30 minutes at 37 ° C with 20 μΙ / well 1% 2 mercaptoethanol (vol / vol) (Bio-Rad, Richmond, CA) to eliminate IgM pentamers. Then, 25 μΙ / well of 10% SRBC suspension was added, followed by 25 μΙ / well of 10 mg / ml (in PBS / 1% glucose) goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL). , as a cross-linking agent for hemagglutination. The titer is defined as reciprocal of the last serum dilution resulting in clear hemagglutination.

Elisha

Plasma mAbs specific for mouse LTB-R (Browning et al., Manuscript in preparation), LFA-3 (Miller, et al., J. Exp. Med., 178, pp. 211 are used for plasma-receptor assays in plasma. -222, (1993)), or the CH3 domain of human IgG (CDG5, prepared at Biogen) directly immobilized (10 μρ / ml) in 96 well microtiter plates for capture and development, donkey anti-human IgG1-horseradish peroxidase (HRP) (Jackson Immuno Research, West Grove PA, 1: 4000 dilution).

Preparation of Soluble LTB-R Molecules

In one embodiment of this invention, LTB-R blocking agents include soluble LTB receptor molecules. Figure 1 shows the sequence of the extracellular portion of human LTB-R that encodes a ligand binding domain. Using the sequence information of Figure 1 and recombinant DNA technologies well known to those skilled in the art, they can be cloned into vector functional fragments encoding the LTBR ligand binding domain and expressed in a suitable host to obtain soluble LTB- R molecule. Soluble LTB-R molecules that can compete with native LTB receptors for binding to the LT ligand according to the methods described in the accompanying pending US Serial Number 08/505, 506, filed July 21, 1995, are selected as LTB- R blocking agents.

A soluble LTB receptor, including amino acid sequences selected from those shown in Figure 1, can be attached to one or more heterologous protein domains ("hybrid domain") to enhance the in vivo stability of the receptor fusion protein or to its biological activity or localization is modulated.

Mostly stable, plasma proteins are used - _? which definitely have a half-life when circulating for more than 20 hours to construct receptor fusion proteins. Such plasma proteins include, but are not limited to:

immunoglobulins, serum albumin, lipoproteins, apolipoproteins and transferrin. Sequences that can capture the soluble LTB-R molecule to a single cell or tissue type may also be attached to the LTB-R ligand binding domain to create a specifically localized soluble LTB-R fusion protein.

The entire or functional portion of the LTB-R extracellular region (Figure 1), including the LTB-R ligand binding domain, can bind to an immunoglobulin constant region, such as the human IgG1 heavy chain Fc domain. (Browning et al., J. Immunol., 154, pp. 33-46, (1995)). Are preferably soluble receptor-lgG fusion proteins, and are common immunological _reagents} and methods for their construction are known in the art (see for example, United States Patent No. 5,225,538 incorporated herein by reference).

A functional LTB-R ligand binding domain can be fused to an immunoglobulin (Ig) Fc domain derived from an immunoglobulin class or subclass other than IgG1. The fc domains of antibodies belonging to different Ig classes or subclasses may activate different secondary effector functions. Activation occurs when the Fc domain binds to a related Fc receptor. Secondary effector functions include the ability to activate the complement system to cross the placenta and bind different microbial proteins. The properties of the different classes and subclasses of immunoglobulins have been described by Roitt et al., Immunology, p. 4.8 (Mosby-Year Book, Europe Ltd., 3d ed., 1993).

Activation of the complement system triggers a cascade of enzymatic reactions that mediate inflammation. The products of the complement system have a variety of functions, including bacterial binding, endocytosis, phagocytosis, cytotoxicity, free radical production and dissolution of immune complexes.

The complementary enzyme cascade can be activated by Fc domains of antigen-bound IgG1, IgG3 and IgM antibodies. The Fc domain of IgG2 appears to be less effective and the Fc domains of IgG4, IgA, IgD and IgE are ineffective at activating complement. Therefore, an Fc domain may be selected based on whether the related secondary effector functions are desirable for a particular immune response or disease treated with the LTB-R fusion protein.

If it is advantageous to damage or kill the LT ligand-bearing cell, a particularly active Fc domain (IgG1) can be selected to produce the LTBR-Ig hybrid protein. Alternatively, if it is desirable to direct the fusion of LTBR-Fc with a cell without initiating the complement system, then an inactive IgG4 Fc domain can be selected.

Mutations in Fc domains have been described that reduce or eliminate Fc receptor binding and complement activation (S. Morrison, Annu. Rev. Immunol., 10, pp. 239-65, (1992)). These or other mutations can be used individually or in combination to optimize the activity of the Fc domain used to construct the hybrid LTB-R-1g protein.

The preparation of a soluble human LTB-R fusion protein comprising ligand-binding sequences fused to the human immunoglobulin Fc domain (hLTB-R-Ig) is described in Example 1. A CHO line was created according to Example 1, which is secreted by hLTB-RFc and named “HLTB-R; hG1 CHO # 14 ”. A sample of this line was deposited on 21 July 1995 at the American Type Culture Collection (ATCC) (Rockville, MD) in accordance with the provisions of the Budapest Treaty and is designated in the ATCC with catalog number CRL11965.

The preparation of a soluble mouse LTB-R hybrid molecule (LTB-R-Ig) is described in Example 2. A CHO line was created according to Example 2, which secretes LTB-R-Ig and is called "mLTB; R-hG1 CHO # 1.3 .VB ”. A sample of this line was deposited on 21 July 1995 at the American Type Culture Collection (ATCC), (Rockville, MD) in accordance with the provisions of the Budapest Treaty and is designated in the ATCC with catalog number CRL11964.

All restrictions on access to the above ATCC deposits will be definitively removed after the patent application has been resolved.

Different amino acid sequences forming the binding portion of the receptor-Ig hybrid protein may alter the structure, stability and basic biological activity of the soluble LTB receptor-hybrid protein. One or more amino acids may be added to the C-terminus of the selected LTB-R fragment to modify the binding site with a selected hybrid domain.

The N-terminus of the hybrid LTB-R protein may also vary by changing the location at which the selected LTB-R DNA fragment breaks at the 5 'end to insert into the recombinant expression vector. The stability and activity of each hybrid LTB-R protein can be analyzed and optimized using routine experiments and assays to select LTB-R blocking agents described herein.

Using LTB-R ligand binding domain sequences in the extracellular domain shown in Figure 1, variants with amino acid sequences can also be constructed to modify the affinity of the soluble LTB receptor or fusion protein to the LT ligand. The soluble LTB-R molecules of this invention can compete with endogenous cell surface LTB receptors for binding to a surface LT ligand. It is contemplated that any soluble molecule comprising the LTB-R ligand binding domain that can compete with cell surface LTB receptors for binding to the LT ligand is an LTB-R blocking agent that falls within the scope of the present invention.

Source of Anti-LTB-R Antibodies

In another embodiment of this invention, antibodies directed against the human LTB receptor (anti-LTB-R Abs) function as LTB-R blocking agents. The anti-LTB-R Abs of this invention can be polyclonal or monoclonal (mAbs) and can be modified to optimize their ability to block LTB-R signaling, their in vivo bioavailability, stability or other desired features.

Serum polyclonal antibodies directed against the human LTB receptor were obtained using conventional techniques by subcutaneous injection of cadavers, goats, rabbits, rats, hamsters or mice, with a human LTB receptor-1g hybrid protein (Example 1), in complete adjuvant Freund, followed by booster intraperitoneal or subcutaneous injection in Freund's incomplete adjuvant. Polyclonal antisera containing the desired antibodies directed against the LTB receptor are screened by conventional immunological procedures.

Mouse monoclonal antibodies (mAbs) were directed against the human LTB receptor-Ig hybrid protein, as described in Applicants Accompanying Application Serial Number 08/505, 506, filed July 21, 1995. A hybridoma cell line was deposited ( BD.A8.AB9), which secretes the mouse anti-human LTB-R mAb BDA8, on January 12, 1995 at the American Type Culture Collection (ATCC) (Rockville, MD) according to the provisions of the Budapest Treaty and is designated in the ATCC with catalog number HB11798. All restrictions on accessibility to the above ATCC deposits will be definitively lifted once the patent application is resolved.

Different forms of anti-LTB-R antibodies can also be obtained using standard recombinant DNA technologies (Winter and Milstein, Nature, 349, pp. 293-99, (1991)). For example, chimeric antibodies may be constructed in which the antigen binding domain of an animal antibody is linked to a human constant domain (e.g., Cabilly et al., US 4,816,667; Morrison et al., Proc. Nat. Acad. Sci., USA, 81, pp. 6851-55, (1984). Chimeric antibodies reduce the observed immunogenic responses elicited by animal antibodies when used in human clinical treatment.

In addition, recombinant "human antibodies" that recognize LTB-R can be synthesized. These "human" antibodies are chimeric, including mainly human IgG sequences into which the regions responsible for specific antigen binding are inserted (e.g., WO94 / 04679). Animals with the desired antigen were immunized, the appropriate antibodies were isolated and the portion of the variable sequence region responsible for specific binding to the antigen was removed. The resulting animal antigen binding regions are then cloned into a suitable site of human antibody genes in which the antigen binding regions are removed. "Human" antibodies minimize the need for the use of heterologous (intraspecific) human antibody sequences and are unlikely to elicit immune responses in treated subjects.

A construct of different classes of recombinant anti-LTB-R antibodies can also be made by creating chimeric or "human" antibodies, including anti-LTB-R variable domains and constant human domains (CH1, CH2, CH3) isolated from different classes of immunoglobulins. For example, anti-LTB-R IgM antibodies with increased valencies to the antigen binding site can be obtained recombinantly by cloning the antigen binding site in vectors carrying constant regions of the human µ chain. (Arulanandam et al., J. Exp. Med., 177, pp. 1439-50, (1993); Lane et al., Eur. J. Immunol., 22, pp. 2573-78, (1993); Traunecker et al., Nature, 339, pp. 68-70, (1989).

In addition, standard recombinant DNA technologies can be used to alter the affinity of binding recombinant antibodies to their antigens by altering amino acid residues near antigen binding sites. The antigen binding affinity of the "human" antibody can be enhanced by mutagenesis based on molecular modeling (Queen et al., Proc. Natl. Acad. Sci .. US, 86, pp. 10029-33, (1989); WO 94 / 04679).

It may be desirable to increase or decrease the affinity of anti-LTB-R Abs to LTB-R depending on the type of stretch considered or the specific treatment program provided. For example, it may be advantageous for the patient to be treated with constant levels of anti LTB-R Abs, with reduced signaling ability via the LT-B pathway, for semi-prophylactic treatments. Similarly, inhibitors of LTB-R Abs with increased affinity for LTB-R are useful for short-term treatment.

Source of Anti-Surface LT Ligand Antibodies

Another preferred embodiment of this invention includes compositions and methods that comprise antibodies directed against the LT ligand that function as LTB-R blocking agents. As described above _} for anti-LTB-R Abs. anti-LT ligand antibodies that function as LTB-R blocking agents can be polyclonal or monoclonal and can be modified according to routine procedures to modulate their antigen binding properties and their immunogenicity.

The anti-LT antibodies of this invention can be generated individually against one of the two LT subunits, including soluble, mutant, altered and chimeric forms of the LT subunit. If LT subunits are used as antigen, it is preferable that they be LTB subunits. If LTa subunits are used, it is preferable that the resulting anti-LTa antibodies bind to the surface LT ligand and do not cross-react with secreted LTa or not modulate TNF-R activity (according to the methods reported in the accompanying US patent application, Serial No. 08/505, 506, filed July 21, 1995).

Alternatively, antibodies against homomeric (LTB) or heteromeric (LTa / β) complexes may be generated that include one or more LT subunits and be screened for activity as LTB-R blocking agents. LTa1 / B2 complexes are predominantly used as antigen. As discussed above, it is preferable that the anti-Ta1 / B2 antibodies obtained bind to the surface LT ligand but do not bind to the secreted LTa and have no effect on TNF-R activity.

The production of polyclonal anti-human LTa antibodies is described in the accompanying pending application of Applicants (WO 94/13808). Monoclonal anti-LTa and antiLTB antibodies have also been described (Browning et al., J. Immunol. 154, pp. 33-46, (1995)).

Mouse anti-human LTB mAbs were obtained as described in the Applicant Accompanying US Application Serial Number 08/505, 506, filed July 21, 1995. A hybridoma cell line was deposited on July 21, 1995 (B9.C9.1) , which secretes murine anti-human LTB-R mAb, B9 at the American Type Culture Collection (ATCC) (Rockville, MD) according to the provisions of the Budapest Treaty and is designated in ATCC with catalog number HB11962.

They were obtained in hamster monoclonal anti-mouse LTo / B antibodies, as described in the Applicant Companion Pending US Serial Number 08/505, 506, filed July 21, 1995. Hybridoma cell line deposited (BB.F6.1 ), which secretes hamster antinish LTa / B mAb BB.F6. on July 21, 1995 at the American Type Culture Collection (ATCC) (Rockviile, MD) under the provisions of the Budapest Treaty and is designated in ATCC with catalog number HB11963.

All accessibility restrictions to upper ATCC deposits will be permanently eliminated after the solution on the patent application. Use of soluble LTB-R-Ig for suppression on

immunological functions of the surface LT complex

We will now show the effects of surface LT binding reagent, a hybrid protein spanning the extracellular domain of murine LTB-R and hinge CH2 and CH3 domains of human IgG1 (LTB-R-Ig), on the origin and nature of the immune responses, on the maintenance of immunological cellular responses organization of secondary lymphoid tissues, including effects on the differentiated state of follicular dendritic cells and the formation of germinal centers and on the expression levels of adrenine that affect cell movement.

Repeated injection of LTB-R-Ig mice alters the organization of splenic lymphocytes and the expression of functional cell markers from the marginal area of the spleen

The effect of surface LT blockade on the structure of the spleen was examined by injecting mice with LTB-R-Ig, 6 times a week. The mice were then immunized with SRBC and injected with LTB-R-Ig 4 days later. Mice were killed 10 days after injection with SRBC. Immunohistochemical staining of frozen sections of the spleen shows some histological changes. Follicles that span the B-cell compartment from the spleen in normal mice are no longer separated after treatment with LTB-R-Ig. In fact, the B cells are now organized in a diffuse band surrounding the T cell regions (Figure 2B) and the border between the T and B cell zones is broken (Figure 2B). In contrast, in control LFA-3-Ig treated mice, B-cell spleen follicles were separated and there was a clear border between T and B-cell regions (Figure 2A).

There is no expression of cell surface markers recognized by the monoclonal antibodies ER-TR-9 and MOMA-1 in two separate macrophage populations inhabiting the marginal area of the spleen of LTB-R-Ig treated mice. ER-TR-9 is known to stain a marker on MZM (Dijkstra et al., Immunol., 55, 23-30, (1985)), and MOMA-1 stains a marker on metalophilic macrophages (Kraal and Janse, Immunol., 58, 665-669, (1986)), (Figures 2 D, F respectively). These markers were expressed on cells in control (LFA-3-Ig) treated mice (Figures 2C, E). Also, LTB-R-Ig treated mice lacked expression of sialoadhesin, another marker of M0MA-1 + macrophages in the marginal area of the mouse spleen (data not shown).

The MESA-367 antibody binds to the adhesive molecule and to the mucous addressin MAdCAM-1. originally described on endothelial cells in Pyer's plaques, mesenteric lymph nodes, intestinal mucosa, and lamina propria (Briskin et al., Nature, 363, pp. 461-464, (1993)); Nakache et al., Nature. 337, p. 179-181 (1989). Expression of MAdCAM-1 has also been described in the marginal area of the spleen (possibly expressed on endothelial cells of the small terminal arterioles opening in the marginal sinus) and on the reticular network in the germinal centers (Kraal et al., Am. J. Pathol., 147, pp. 763-771, (1995)), (Figure 2 G). MECA-367 stained sections of LTB-R-Ig treated mice indicate that MAdCAM-1 expression is destroyed in the spleen (Figure 2 H).

Similarly, staining with an ER-TR-7 antibody (Van Vliet et al., Cytochem., 34, pp. 883-890, (1986)), which outlines a population of reticular fibroblasts in the marginal area (Figure 2 I), is abnormal. is distributed and is stronger in the white pulp of LTB-R-Ig treated animals than in those treated with LFA-3-Ig (Figure 2 J). The changes observed in LTB-R-Ig treated mice are not dependent on antigen exposure, since the staining pattern is identical in LTB-R-Ig treated mice (data not shown).

Formation of germinal center is prevented and no follicular dendritic cells are detected in the spleens of LTB-R-Ig treated mice

To determine histologically how multiple injection of LTB-R-1g mice affects the immune response to SRBC, germinal center formation (GC) and follicular dendritic cell division (FDC) analysis were performed. exposure with antigen. Frozen slices of mouse spleen, pre-treated with LTB-R-Ig or LFA-3-Ig, as described in Figure 2, were stained with peanut agglutinin (PNA) to delineate GCs and the FDC- antibody M1, for the detection of FDC, a cellular component required for the formation of GC (Schriever and Nadler, Adv. Immunol., 51, pp. 243-284, (1992)); Tew et al., Immunol. Rev., 117, pp. 185-211 (1990). It has also been shown that the CD40-CD40 ligand interaction is also critical for GC formation (Foy et al., J. Exp. Med., 180, pp. 157-163, (1994)). For comparison, a group of mice with MR1, an anti-mouse CD40 ligand antibody, were treated according to the injection protocol, which was previously shown to inhibit GC formation (Han et al., J. Immunol., 155, pp. 556-567 , (1995)). Ten days after immunization with SRBC mice treated with control LFA-3-Ig protein developed a large number of PNA bright GCs in the spleen (Figure 3 A). GCs were not detected in the spleen of LTB-R-Ig or MR1-treated mice (Figures 3 B, C respectively). However, the effect of MR1 and LTB-R-Ig can be distinguished by two additional observations. Staining for FDCs (FDC-M1) in GC (Figure 3 D) is absent in the spleen of LTB-R-Ig mice (Figure 3 E), but still observed in the spleen of MR1 treated mice (Figure 3 F) ). Similar observations were made using the FDC-M2 antibody to stain FDCs (data not shown). Therefore, treatment with LTB-R-Ig results in a phenotypic alteration of FDC in the spleen and inability to form GC.

In addition to staining GC, PNA also stains the marginal area in the spleen of normal mice. Such staining was also noted in mice treated with LFA-3-Ig (Figure 3 A) and MR1 (Figure 3 C), but was absent in the spleen of LTBR-Ig treated mice (Figure 3 B).

The expression of siapoadhesin, MOMA1, ER-TR-9, ER-TR-7 and MAdCAM-1 was also shown to be normal in the spleen of MR1-treated mice (data not shown), which further leads to distinguishing between molecular effects of CD40 interference and LTB-R signaling.

Kinetics of LTB-R-Ig induced changes in splenic lymphocyte organization and expression of cellular markers from the marginal zone

The number of LTB-R-Ig injections required to affect the lymphocyte organization and expression of cell markers from the marginal area in the spleen was analyzed. Ip mice were injected with LTB-R-Ig once or repeatedly, as shown in Table 1. Thereafter, some mice were immunized with SRBC on the day of the last LTB-R-Ig injection. The expression of B220 and CD4 on Vi T cells ₇ and staining with PNA (for GC) and MECA-367 (for MAdCAM-1), MOMA-1, ER-TR-9 and FDC-M1 were determined on frozen sections of spleen of treated mice. The disappearance kinetics of staining of metalophilic macrophages, marginal zone macrophages, MAdCAM-1, GCs and FDCs have been shown to be different.

One injection of LTB-R-Ig is sufficient to eliminate one week later staining with MAdCAM-1. No staining of GCs and FDCs was detected at the subsequent three LTB-R-IG injections per week, and T / B lymphocyte compartments were destroyed. A minimum of four LTB-R-Ig injections are required to eliminate staining of metalphilic macrophages. Six LTB-R-Ig injections do not completely eliminate macrophage staining from the marginal area with ER-TR-9 antibody (also shown in Figure 2 D).

More precise analysis was performed in the absence of antigen to rapidly suppress the staining of MOMA-1, MAdCAM-1, FDC-M1, FDCM2 and CR1 after a single injection with LTB-R-Ig (Table 2)

not done

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Table 2: Precise determination of the effects of LTB-R-Ig on the staining of MOMA-1, MAdCAM-1, CR1, FDC-M1 and FDC-M2

DAYS AFTER SINGLE INJECTION With LTB-R-lg

0 1 3 5 7 10 14 MOMA-1 * +++ +++ +++ +++ t + ++ + MAdCAM-1 +++ +/- - - - - - CR1 * +++ +++ ++ ++ J- + ++ + FDC-M1 +++ +/- - - - - - FDC-M2 +++ +/- _β

Baib / c mice, 5-6 weeks old, received one ip injection of 100 µg LTB-R-Ig or human Ig. Mice from each group were killed on days 0, 1, 3, 5, 7, 10 and 14. Frozen spleen sections were stained with the following antibodies: rat anti-mouse metallophilic macrophages (MOMA-1), rat anti-mouse or MAdCAM-1 (MESA 367 ), rat anti-mouse FDC (FDC-M1), rat anti-mouse FDC (FDC-M2), and biotin-labeled rat anti-mouse CR1, followed by mouse anti-rat lg peroxidase (MOMA-1, MAdCAM-1, FDC-M1 FDC-M2 ) or peroxidase-labeled streptavidin (CR1).

* The intensity of coloration is determined by eye: normally colored: +++, less colored: ++, slightly colored: + and unstained: -. The intensity of staining on sections of untreated animals and animals treated with human Ig was taken as the standard for normal staining. Sections of two animals from at least a group were analyzed.

Balb / c mice given a single ip injection of LTB-R-lg _? are killed daily, 14 days after the injection and their spleens are removed and frozen. Frozen spleen sections were stained with MOMA-1, anti-MAdCAM-1 (MECA-367) and FDC specific reagents: FDC-M1, FDC-M2 and anti-CRI antibodies. One day after LTB-R-Ig injection, staining with anti-MAdCAM-1, FDC-M1 and FDC-M2 reagents decreased very strongly (Table 2). The rapid suppression of FDC-M1 staining in this experiment compared to the results ₇ described in Table 1 may be due to the intensity of FDC-M1 staining, which is stronger in immunized animals. CR1 staining was still detected at day 14, indicating that FDCs were still present on day 3 after LTB-R-Ig treatment, but that expression of markers detected with FDC-M1 and FDC-M2 was suppressed. Therefore, treatment with LTB-R-Ig changes the FDC phenotype. Finally, MOMA-1 staining was reduced, but was still detectable at day 14.

Repeated treatment with LTB-R-Ig suppresses the expression of adrenine in the LN.

The expression of adrenine in LN in progeny of pregnant Balb / c mice that were injected at day 14 and 17 of pregnancy with 200 µg receptor Ig proteins was examined. After birth, is the generation not treated _? or injected once a week with 100 µg LTB-R-Ig, TNF-R55-Ig or LFA-3-Ig. As long as the generation is alive, the levels of the hybrid protein remain up to or about 10 µg / ml, which are determined by ELISA (data not shown). Immunohistochemical staining with MECA367 and MECA79 shows that MAdCAM-1 and peripheral LN addresses are completely missing in mesenteric LN from treated mice _? while alive with LTB-R-Ig (Figure 4 A, B). The sacral LN of these mice also lacked expression of all the adrenaline, and the cervical and those of the groin LN did not show staining of the peripheral lymph nodes (PNAd values not shown). Reduced expression of addressins is reversible as expression is restored to normal levels in animals treated only in utero (Figure 4 G, H). In mice treated while alive at 100 µg / week with TNF-R55-Ig or LFA-349 Ig, expression of adrenaline in LN remained comparable to untreated mice (Figure 4 C, D, E. F).

B-lymphocyte positioning and macrophage marker expression is altered in the LN of LTB-R-Ig treated mice.

Antibodies are used that bind markers to macrophage populations in the LN subcapsulated sinus (analogue of the marginal area of the spleen) to perform immunohistochemical analyzes of L Ν, β3βτ from mice treated during pregnancy and continuously after birth, continuously after birth. as described in Figure 4 above with LTB-R-Ig, TNF-R55-Ig or LFA-3-Ig. Fluorescence imaging was analyzed using software imaging assays. The expression of sialoadhesin was shown to decrease in the LN of mice treated with soluble LTB-R-Ig (Figure 5 B) but not in the LN of mice treated with TNF-R55-Ig or LFA-3-Ig (Figure 5B). 5 E, H). The expression of MOMA-1 on macrophages in the subcapsular sinus in the LN of LTBR-1g treated mice is still detected (Figure 5 C).

The effects of continuous LTB-R-Ig treatment on the organization of lymphocytes in the LN are also evaluated. LN sections were stained with mAbs specific for the B-cell marker B220 and the T-cell marker CD4. Imaging assays are used to identify areas of overlap of T and B cell zones. Treatment with LTB-R-Ig results in the disintegration of B-cell follicles such that the B-cells are present as a diffuse band on the outer end of the T-cell region (Figure 5 A). Despite the breakdown of their follicular structure, B cells are not present in the T cell regions of the LN, instead they appear in areas that are not normally occupied by lymphocytes. A very similar pattern of T and B cell staining is observed in mice treated while alive _? with 100 µg / week of TNF-R55-Ig but not with LFA-3-Ig (Figure 5 D). Again, B-cell follicles are disrupted and B-cells are present in areas of LN that usually do not contain lymphocytes. No overlap of B with Tcells was observed.

Treatment of LTB-R-Ig mice inhibited IgM and IgG responses

The inability to form GC in spleen after SRBC priming of LTB-R-Ig-treated mice repeatedly (as in Figure 3) allows changes in the humoral immune response of these mice. To check this directly, adult mice received six injections of LTB-R-Ig or LFA-3-Ig once a week and then primed with SRBC. The mice were then bled at 7 and 14 days after immunization and the presence of SRBCspecific IgM and IgG in the serum was analyzed using hemagglutinin analysis. Seven days after SRBC immunization, the IgM titer was normal, but the IgG response in LTBR-Ig treated mice was largely suppressed compared to those treated with human Ig or PBS. (Figure 6 A). On day 14 after immunization, no SRBC specific IgG was detected in the serum of LTB-R-Ig mice treated and the SRBC specific IgM titer in these mice also decreased by more than half compared to human Ig or PBS treated mice ( Figure 6 A).

Ten days after SRBC priming, GCs were detected in the spleens of mice treated once or twice with LTB-R-Ig, however, the number of GCs was greatly reduced compared to controls (Table 1). When mice received two injections of LTB-R-Ig, the first injection 1 week before SRBC priming and the second injection on the same day of SRBC injection, the suppression of IgM and IgG response to SRBC at days 7 and 14 (Figure 6 B) was similar with that obtained when mice received repeated LTB-R-Ig injections (Figure 6 A). At day 30 after immunization, no SRBC-specific IgG was detected and IgM levels were reduced by more than 80% compared to controls (Figure 6 B). Therefore, treatment with LTB-R-Ig results in complete suppression of IgG responses and a reduced / suppressed IgM response similar to controls.

When mice received a single injection of LTB-R-ig, on the day of SRBC priming, the levels of IgG and IgM responses to SRBC at day 7 were comparable to those of the control group (Figure 6 C). However, at day 24 after immunization, the IgM and IgG titers were reduced by 30%. At 34 days after SRBC priming, the titer of SRBC-specific IgM was reduced by 50% compared to controls, and SRBC-specific IgG could not be detected (Figure 6 C). These data indicate that treatment with LTB-R-Ig leads to a significant decrease / reduction in levels of both IgM and IgG responses, which means that LTB-R-Ig treatment can suppress the humoral response, which is already initiated.

Mediated Disease Antibody

Many cases of autoimmune diseases, organ-specific and multisystemic, involve pathological antibody responses. These cases include: Myasthenia Gravis, autoimmune hemolytic anemia, Chagas 'disease, Graves' disease, idiopathic thrombocytopenia purpura (ITP), Systemic Lupus Erythematosus (SLE), Wegener's Granulomatosis, Poly-arteritis Nodosa, and Rapidly Progressive Glomeritis. (From Benjamini, et al., Immunology, A Short Course, (Wiley-Liss, New York, 3d ed., (1996)).

Although the etiology of SLE has not been defined, there is satisfactory evidence of the immunological mechanism responsible for the pathology observed. For unknown reasons, patients with SLE produce antibodies against the nuclear components of the body (ANA), mainly against native, double stranded DNA. Clinically, the presence of these antibodies correlates best with the kidney pathology involved in SLE. with DNA probably derived from damage to normal tissue and as in any disease of immune aggregates, these complexes form deposits that localize to the basement membrane of the glomeruli, the arterial walls, and the synovial joints. These complexes activate the complement cascade and attract granulocytes, and subsequently the inflammatory response is characterized as glomerulonephritis, which as a result damages the kidneys and thus leads to proteinuria and hematuria.

Lupus nephritis has been studied in mouse models for decades. Recently, the therapeutic effect of a mouse CD40 specific ligand specific reagent was evaluated in such a model (Mohan, et al., J. Immunol. 154, pp. 1470-1480 (1995)). In vivo, it has been shown that the increase in lupus by transferring cells that induce pathogenic antibodies is inhibited by the introduction of a monoclonal antibody that blocks ligand CD40 / CD40 interactions. Furthermore, short-term treatment of mice suffering from lupus with an antiCD40 ligand antibody has proven to have a beneficial effect on their spontaneous disease long after the body has been cleared of the antibody. The experiment shows that pathogenic Cells may not produce an antibody even 9 months after therapy, suggesting that there is a delay in the expansion of autoimmune B cell memory, leading to a long lasting therapeutically beneficial effect. As we have shown, these reagents, which block LTcdVLTB-R interactions in vivo, suppress the emergence of antibody responses, alter the FDC phenotype and the formation of germinal centers involved in the optimal emergence of B-cell memory. The blocking LTcxB / LTB-R reagents of this invention will be useful for the treatment and prevention of SLE.

Normal immune responses to some pathogenic infectious agents also elicit autoantibody responses that are excess and a medical problem. One such example is Chagas' disease, an inflammatory cardiomyopathy that develops in humans and experimental animals with chronic Trypanosoma cruzi infection. Among the possible mechanisms involved in the pathogenesis of human Chagas' cardiomyopathy, significant experimental confirmation of the induction of cardio-specific autoimmune responses has recently been obtained. A modern study (Tibbetts, et al., J. Immunol. 152, pp. 1493-1499, (1994)) determined that cardiac antigen-specific antibodies were produced in the. T. Cruzi - infected, C57B1 / 6 mice with heart disease. In infection with a Brazilian strain of T. Cruzi, C57B1 / 6 mice develop cardiomyopathy, which is histologically similar to that seen in chronically infected humans. Antisera from these mice interact with three cardiac antigens, whereas C57B1 / 6 mice infected with T. Cruzi's Guayas strain that do not develop cardiomyopathy do not produce such antibodies. These data indicate that these antibodies are specific markers for cardiomyopathy. Therefore, using such a model, the ability of LTB-R blocking agents to suppress autoantibody-mediated damage can be evaluated.

Another example of cellular damage by autoantibodies produced due to certain infectious diseases or for other unknown reasons is idiopathic thrombocytopenia purpura (ITP). In this case, antibodies directed against platelets lead to the destruction of the platelets (via complement or phagocytic cells with Fc or C3b receptor), which in turn leads to hemorrhage. Therapists who will suppress such an antibody mediating autoimmune reactions in vivo, such as the LT-B-R blocking agents of this invention - - which inhibit antibody formation - - will be useful for both treatment and prevention of these autoimmune diseases.

The normal immune response to some pathogenic infectious agents also triggers hypersensitivity reactions, which can become a threat and in itself a medical problem. The most common example of type I hypersensitivity is allergic reactions. They are mediated by IgE antibodies that, through their Fc moiety, bind to mast cells and basophils to unlock the release of pharmacologically active anaphylaxis mediating agents. ITP and Goodpasture syndrome are thought to be type II reactions that occur when IgM and IgG antibodies bind to the cell surface antigen and activate the complement cascade. The granulocytes are then attracted to the site of activation and the damage resulting from the release of lytic enzymes from their granules leads to cell destruction. Rheumatoid arthritis is thought to lead to a type III hypersensitivity reaction mediated by immune complexes of antigen (in this case rheumatoid factor, IgM autoantibody) that binds to the Fc portion of normal IgG. These immune complexes cause inflammation of the joints and typical damage to the disease. Because pathological conditions are partially mediated by antibodies, therapists who will suppress antibody formation, such as the LTB-R blocking agents of this invention - will be useful for both treating and preventing these diseases.

Treatments by Using LTB-R Blocking Agents

The compositions of this invention should be administered as an effective dose to treat each individual case. The determination of the preferred pharmaceutical composition and the therapeutically effective dose for a given administration is well known to one of skill in the art, given, for example, the case and weight of the patient, the extent of the desired treatment and the patient's tolerance for treatment.

Doses of about 1 mg / kg of soluble LTB-R are thought to be suitable as a starting point for optimizing treatment doses.

Determination of a therapeutically effective dose can also be assessed by conducting in vitro experiments by determining the concentration of the blocking LTB-R agent required to cover the target cells (LTB-R or LT ligand-positive cells relative to the blocking agent) from 1 to 14 days. The previously described receptor-ligand binding methods, in the Applicant's pending US patent application Serial Number 08/505, 506, filed July 21, 1995, can be used to control the cell coverage response. Using FACS, ligand-positive LTB-R or LT cells can be separated from activated lymphocyte populations. Based on the results of in vitro binding methods, suitable concentrations of LTB-R blocking agents can be selected for testing in animals.

The introduction of soluble LTB-R molecules, anti-LT ligand and anti-LTB-R Abs of this invention, alone or in combination, including isolated or purified forms of antibodies or complexes, their salt or pharmaceutically acceptable derivatives, can be accomplished by any of the conventional methods of administering agents that exhibit immunosuppressive activity.

The pharmaceutical compositions used in these therapies may take various forms. For example, they include doses of solid, semi-solid and liquid forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories and injectable and infusion solutions. The appropriate formulation is determined by the preferred route of administration and therapeutic administration. Administration may include, orally, parenterally, subcutaneously, intravenously, intralesional (in injured tissue), or topical administration.

The soluble LTB-R molecules, anti-LT ligand, and anti-LTB-R Abs of this invention can be placed, for example, in sterile isotonic mixtures with or without cofactors that stimulate absorption or stabilization. Preferably, the composition is liquid or in the form of a lyophilized powder. For example, soluble LTB-R molecules, the anti-LT ligand, and anti-LTB-R Abs of this invention can be dissolved in mixed buffer containing 5.0 mg / ml acetic monohydrate. 2.7 mg / ml trisodium citrate, 41 mg / ml mannitol, 1 mg / ml glycine and 1 mg / ml polysorbate 20. This solution may be lyophilized, stored in the cold and, before administration, prepared with sterile water for injection. (USP).

Is preferably also mixtures include conventional pharmaceutically acceptable carriers well known in the art (see for example, Remington's Pharmaceutical Sciences, 16 ^th Edition, 1980, Mac Publishing Company). Such pharmaceutically acceptable carriers may include other therapeutic agents, carriers, genetic carriers, adjuvants, excipients, and the like, such as human serum albumin or plasma preparations. Preferably, the compositions are in unit dosage form and are usually administered once or more times daily.

The pharmaceutical compositions of this invention may also be administered using microspheres, liposomes, or other microcarrier systems, or confirmed systems, delivering and releasing the mixtures incorporated therein at a precise location, in proximity or elsewhere for interaction with damaged tissues or in the blood stream. Suitable examples of such stable carriers include semipermeable polymer matrices in the form of certain articles such as suppositories or microcapsules. Such implants that can be implanted or are resistant as microcarriers include polylactides (US Patent No. 3,773,319; EP 58,481), copolymers of L-glutamic acid and ethyl-L-glutamate (Sidman et al. Biopolymers, 22, pp. 547-56, (1985)); poly (2-hydroxyethyl methacrylate) or ethylene vinyl acetate (Langer et al., J. Biomed. Mater. Res., 15, pp. 167277 (1981); Langer, Chem. Tech., 12, pp. 98-105 (1982 )).

Liposomes containing soluble LTB-R molecules, anti-LT ligand and antiLTB-R Abs of this invention can be prepared individually or in combination by well known methods (See, e.g., DE, 3, 218, 121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82, pp. 3688-92, (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77, pp. 4030-34, (1980); US Patent Nos. 4, 485, 045 and 4, 544, 545). Usually liposomes are small (about 200-800 Angstroms), single-membrane, in which the lipid content of cholesterol is more than 30 mol. %. The cholesterol proportion is selected to control the optimum release rate of soluble LTB-R molecule, anti-LT ligand and anti-LTB-R Ab.

The soluble LTB-R molecules, anti-LT ligand, and anti-LTBR Abs of this invention can also be attached to liposomes containing other LTB-R blocking agents, immunosuppressive agents or cytokines to modulate blocking LTB-R activity. The attachment of LTB-R molecules, anti-LT ligand and anti-LTB-R Abs to liposomes can be accomplished through any known cross-linking agent, for example as heterobifunctional cross-linking agents that are widely used to bind toxins or chemotherapeutic agents with antibodies to get to the right place. Conjugation with liposomes can be accomplished by using a carbohydrate cross-reacting 4- (4-maleimidophenyl) butyric acid (MPVH) hydrazide (Duzgunes et al., J. Cell. Biochem. Abst. Suppl., 16E , 77, (1992).

Advantages of therapeutic mixtures containing LTB-R blocking agents.

The LTB-R blocking agents of this invention are capable of selectively suppressing immune effector mechanisms. Antibody-mediated immunity-mediated inhibition is inhibited by a number of mechanisms involving the regulation of GC formation by affecting FDC function. Both the antibody and the cell-mediated immunity are partially suppressed by regulating the expression of the adrenals and thus affecting the movement of lymphocytes. Therefore, LTB-R blocking agents will be useful in treating cases that are exacerbated by antibody activity or abnormal expression of addressins. The ability to selectively suppress these immune-mediated responses is useful for treating various abnormalities involving various autoimmune and chronic inflammatory processes. As discussed above, immunomodulatory immunosuppressive agents are commonly used in the treatment of these pathologically immune-mediated cases, which have pleiotropic effects on many diverse cell types and immunological responses. Usually, these non-specific immunosuppressive agents are required at high and often cytotoxic doses that have other side effects. The ability of the reagent to suppress antibody responses, in order to facilitate the pathological immunological response, is confirmed in the present study of murine lupus nephritis. The latter study showed that administration of an antibody that blocks the CD40 / CD40 pathway suppresses the increase in lupus nephritis produced by the transfer of cells that induce the production of pathogenic antibodies in vivo and has a stable and beneficial effect on spontaneous disease long after the body being cleared of the antibody. These data indicate that the blocking LTB-R agents of this invention can be used to suppress rejection of tissue grafts and organ transplants by inhibiting the processes leading to the emergence of antibody responses.

The LTB-R blocking agents of the mixtures and methods of this invention can be modified to obtain the desired levels of LTB-R signaling, depending on the condition, damage or treatment of the disease. It is contemplated that the absolute level of LTB-R signaling can be regulated by altering the concentration and affinity of LTB-R blocking agents to their molecular targets.

For example, in one embodiment of this invention, mixtures comprising soluble LTB-R molecules are introduced into the site. The soluble LTB receptor can effectively compete with cell surface LTB receptors for binding to surface LT ligands. The ability to compete with surface LT ligands depends on the respective concentrations of soluble and cell surface LTB-R molecules and on their relative affinity for ligand binding.

Soluble storage mutant molecules that increase or decrease the binding affinity of this mutant soluble LTB-R with the surface LT ligand can be obtained using standard recombinant DNA technologies well known to those skilled in the art. Using the routine experiments and techniques described herein, a large number of molecules with specific or random mutations can be tested for their ability to act as LTB-R blocking agents.

Similarly, in another embodiment of this invention, antibodies directed either against the LTB-R receptor or against one or more of the LT ligand subunits function as LTB-R blocking agents. The ability of these antibodies to block LTBR receptor signaling may be modified by mutation, chemical modification, or by other methods that alter the effective concentration or activity of the antibody delivered to the subject.

The ability to attenuate LTB-R signaling without being completely suppressed may be essential for detecting or maintaining low levels of LTB-R signaling that maintain normal immune function by inhibiting antibody or cell-mediated responses that are too strong or anomalous.

Damage to the 1Ta gene in a mouse results in the development of abnormal peripheral lymphoid organs (De Togni et al., Science, 264, pp. 703-707, (1994)). Such mice lack lymph nodes and usually do not clearly detect Τη B-cell-rich areas in the follicles in their spleens. We believe that this phenotype is associated with the loss of surface LT induced LTB-R signaling, because similar phenotypes are not observed when modulating TNF-R activity. The ability to selectively or partially block the LTB-R pathway may be useful in the treatment of abnormally developing lymphoid-like structures as a result of chronic inflammation associated with the absence or overexpression of LTB-R pathway signaling.

Antibodies are important mediators of immune responses to pathological agents. Therefore, absolute suppression of antibody responses may not be desirable in some cases. For example, antibodies are needed to mediate resistance to infections by extracellular bacteria, such as pneumococci and hemophilus. The ability to influence the antibody level due to blocked LTB-R signaling may be important for obtaining the most useful results that can be achieved by treatment with the LTB-R blocking agents of this invention.

The therapeutic methods of the invention include selectively inhibited responses that are wholly or partly dependent on the LT-β pathway. The specific therapeutic benefits of the invention depend on the appropriate etiological mechanism, whether the process is suppressed or, if necessary for treatment, aided, which is clear to those skilled in the art. Therefore, the methods of the invention include various variants, the introduction of a therapeutically effective amount of a blocking agent of LT-B-R or LT-β. The protein used in these methods may be either full-length protein, protein fragments or hybrid fragments. In other embodiments, methods include introducing a soluble fragment, for example soluble lymphotoxin-β receptor. Other preferred embodiments of the invention pertain to the introduction of antibodies against LT-B-R or LT-β. The blocking agents of the invention can be administered competitively with a therapeutically effective amount of a second compound that enhances the desired therapeutic effect.

For example, in some methods of treating spin and / or HIV, it may be desirable to introduce at the same time additional antiviral agents known to those skilled in the art. For example, AZT or protease inhibitors. Particularly preferred is the administration of blocking agents of the invention, more preferably, the LT ^ -R / IgG hybrid protein in combination with spin "cocktail" therapy. These drug "cocktails" include the introduction into the patient of many drugs to reduce the amount of virus in the patient's body.

The mixtures of the invention may be formulated in accordance with standard practice, for example as prepared in a carrier. The term pharmaceutically acceptable carrier refers to one or more organic or inorganic ingredients, natural or synthetic, which may facilitate the administration of the blocking agents of the invention to the patient. Suitable carriers are well known in the art.

Each of the compositions of this invention can be administered in any way as long as it is acceptable for treatment. This includes injections via the parenteral route, such as intravenous, intravascular, intra-arterial, subcutaneous, intramuscular, intratumoral, intraperitoneal, intraventricular, intraepidural or other, as well as oral, nasal ocular, rectal or topical. The invention specifically includes confirmed carriers for administration by depot injection or implant. Accurate delivery and local release may also be desirable. Methods of administration are readily determined by those skilled in the art.

The LT pathway blocking agents used in the invention are selected to include functionally derived * soluble LT-B-R antibodies, the antibodies disclosed herein. Functionally derived include fragments, variants, analogs or chemical derivatives of a molecule. A fragment of a molecule, considering any of the antigens of the present invention, pertaining to any polypeptide portion of the molecule. An embodiment of such a molecule means to refer to a naturally occurring molecule substantially similar to the whole molecule or to a fragment of it. An analog of a molecule refers to an unnatural molecule that is substantially similar to the whole molecule or to a fragment of it.

The variants of the blocking agents of the invention differ from naturally occurring agents in amino acid sequence or in the non-sequencing manner, or both. Variants in amino acid sequence are obtained when one or more amino acids in naturally occurring molecules are replaced by another natural amino acid, derived amino acid or unnatural amino acid. Particularly preferred variants include naturally occurring proteins or biologically active fragments of naturally occurring proteins whose sequence differs from a given sequence by one or more conservative amino acid substitutions. These substitutions are well known to those skilled in the art and generally have minimal effect on the secondary structure and hydrophobic nature of the blocking agent.

In other embodiments, variants with amino acid substitutions that are less conservative may also produce the desired derivatives, i.e. by changing charge, conformation or other biological properties. For example, such substitutions may include replacement of hydrophilic residues with hydrophobic residues, replacement of cysteine or proline with another residue, replacement of a residue having a small side amino acid chain with a residue having a long side chain, or replacement of a positive charge with a residual charge. Where the result of a substitution cannot be predicted with certainty, the derivatives may be immediately analyzed according to the methods set forth herein for determining the presence or absence of the desired characteristics.

Variants within the scope of the invention include proteins and peptides with amino acid sequences having at least eighty percent homology to the blocking agents of the invention. Preferably, the sequence homology is at least ninety percent or at least ninety-five percent. In order to determine the homology, the length of the sequences compared must be at least 8 amino acid residues, usually at least 20 amino acid residues. Embodiments of the invention also include any blocking agent that 1) has an amino acid sequence that is at least forty percent homologous to the blocking agent sequence and which also 2) is positioned optimally in a straight line with respect to the sequence of the invention. the blocking agent of the invention, its cysteine residues to coincide with at least 80% of the cysteine residues of the blocking agent of the invention.

There are also agents in the invention that bind specifically to the blocking agents of the invention, including ligands and antibodies.

The following are examples to illustrate soluble LTB receptors, anti-LT ligands, and anti-LTB-R antibodies of this invention and methods used to characterize them. These examples are not to be construed as limiting, they are incorporated by way of illustration and the present invention is only limited by the claims.

EXAMPLES FOR THE IMPLEMENTATION OF THE INVENTION

EXAMPLE 1

Preparation of soluble human LTB-R receptors such as immunoglobulin Fc fusion proteins

A sequence of a human cDNA clone isolated from a library of human 12p transcribed sequences derived from a somatic cell hybrid (Baens et al., Genomics, 16, pp. 214-218, (1993)) was recorded in GenBank and was later identified as a sequence encoding human LTB-R. The full sequence of this human LTB-R cDNA clone has been available since 1992, registered with GenBank with L04270.

The extracellular domain of LTB-R to the transmembrane portion (Figure 1) is amplified by PCR from a cDNA clone, using όό primers that insert Not I and Sal I restriction sites at the 5 'and 3' ends respectively (Browning et al J. Immunol., 154, pp. 33-46, (1995). The amplified product was cut with Not I and Sal I, purified and ligated into the Not l-lianised vector pMDR901, together with the Sal l-Not I fragment. encoding the FC region of human lgG1. The resulting vector contains a dihydrofolate reductase gene and an LTB-R-Ig hybrid protein, driven by individual promoters.

The vector is electroporated into CHO dhfr cells and methotrexate-resistant clones isolated according to standard procedures. LTB-R-Ig is secreted into the medium and an ELISA method is used to select the cell lines producing the highest levels of the receptor fusion protein. The strongest producing cell line is grown in large quantities and in a suitable environment. Pure LTB receptor fusion protein was isolated by Protein A Sepharose Fast Flow Affinity Chromatography (Pharmacia).

EXAMPLE 2

Preparation of Soluble Mouse LTB Receptors as Immunoglobulin Hybrid Proteins

A complete LTB-R mouse cDNA clone was prepared by ligation of 5 'Not l / Apal I and 3' Apal l / Not I fragments of two partial cDNAs isolated at the Not I site of pCDNA3 (InVitrogen, San Diego, CA). The sequence of this cDNA clone is available and registered in GenBank with U29173. No differences were observed in the coding sequence when compared with another sequence registered for the murine LTB-R found in GenBank and registered with L38423.

0 /

A soluble mouse LTB-R / human IgG1 hybrid protein was prepared by full-length PCR amplification of mLTB-R cDNA clone as template and primers 5 'AACTGCAGCGGCCGCCATGCGCCTGCCC 3' and 5 'GACTTTGTCGACCGGGGG

The amplified product was purified and cut with Not I and Sal I and ligated with a Sal l / Not I human IgG1 Fc fragment in Not Ilinized and phosphatase-treated SAB132 to form JLB 122. For stable expression, the Not I cassette containing the murine LTB-Rlg fragment was transferred to the Not I site of pMDR901, forming PSH001, and the vector was transferred to CHO cells as described by (Browning et al., J. Immunol, 154, pp. 33-46, (1995) ). Cell clones secreting mouse LTB-R-Ig will be identified by ELISA analysis. The purified receptor fusion protein was isolated from CHO cell supernatants by Protein A Sepharose Fast Flow Chromatography (Pharmacia) and illustrated by the following examples.

EXAMPLE 3

Immunohistochemical analysis of spleen from multiple injections of LTB-R-1g mice

Mice 4-5 weeks old received six injections once a week from LTB-R-Ig or LFA-3-Ig (100 µg ip) and were immunized with SRBC on the day of the sixth hybrid protein injection. The mice were then further injected with LTB-R-1g or LFA-3-Ig, 4 days after immunization with SRBC. At day 10 after immunization with SRBC, the animals are killed and the organs analyzed for structure analysis. The left column of Figure 2 shows slices of spleen of animals treated with LFA-3-ig (A, C, E, G, I) and the right column of animals treated with LTB-R-Ig (B, D, F, H, J). Frozen acetone-fixed slice sections were double-stained with biotinyl pranic anti-mouse labeled B220 and anti-mouse CD4 antibodies (A and B) and then stained with alkaline phosphatase-labeled streptavidin (purple blue, dark colored respectively) horseradish peroxidase - labeled mouse anti-rat Ig (light brown colored). Other frozen sections were stained with ER-TR-9 (for detection of MZM, C and D), MOMA-1 (for detection of macrophages, E and F), MECA-367 (specific for MAdCAM-1, G and H) and ER-TR-7 (for staining of reticular fibroblasts, I and J) antibodies and then peroxidase-stained mouse anti-mouse Ig (brown stained). These photos represent colored sections of at least six animals. Magnification 10x.

EXAMPLE 4

Effect of LTB-R-Ig and anti-C040 ligand on GC formation and FDC staining

Animals were treated as described in Example 3 with LTB-R-Ig or LFA-3-Ig. Another group of animals were treated with MR-1 (anti-mouse CD40 ligand, 250 µg / injection, intraperitoneally) on day -1, day 1 and day 3, received SRBC on day 0 and killed on day 10. Acetone-fixed sections of the spleen of animals treated with LFA-3-Ig (Figure 3 left column, A and D) or. With LTB-R-Ig (middle column, B and E), or MR1 (right column, C and F) if stained with biotin-labeled peanut agglutinin (PNA, top row, A, B and C) or with FDC-M1 (bottom row, D, E and F) followed by a second peroxidase-labeled staining streptavidin and peroxidase-labeled mouse antiglare Ig, respectively (brown colored). PNA staining of the marginal area is indicated by an arrow in A and C. Formation of the GC is indicated by a white asterisk in A. FDC staining is indicated by a black arrow in D and F. These photographs show sections of at least four animals. Magnification 10x.

EXAMPLE 5

Expression of adrenine in LN from mice treated in utero and long after birth with LTB-R-1g

In these experiments, pregnant Balb / c mice were used which were injected iv at 14 and 17 days of gestation with 200 µg of receptor-1g proteins. After birth, the offspring are injected ip with 100 µg LTB-R-Ig, TNF-R55-Ig or LFA-3-Ig. Throughout life, the levels of the hybrid protein determined by E LISA / remain at or above 10 µg / ml (data not shown). Figure 4: Panels A, B, G and Lymph node staining of LTB-R-Ig treated mice. Panels C, D - Lymph node staining of mice treated with LFA-3-Ig; Panels E, F - staining of lymph nodes from mice treated with TNF-R55-Ig. Panels A, C, E, G are mesenteric lymph nodes stained with MESA-367 antibody to detect mucosal addressin, MAdCAM-1. Panels B, D, F, H are brachial lymph nodes stained with MESA-79 antibody specific for peripheral LN addresses (PNAds). Panels G, H are lymph nodes of 6-week-old mice exposed only to LTB-R-Ig in utero. All photos are zoomed 50x.

EXAMPLE 6

Lymphocyte location and expression of macrophage markers in LN from mice treated in utero and long after birth with LTB-R-Ig

Mice were treated in utero and long after birth as described in Example 5 with LTB-R-Ig, TNF-R55-Ig or with LFA-3-Ig. LN sections were then stained with antibodies specific for macrophage-expressed markers or mAbs specific for the B cell marker B220 and the T cell marker CD4. Imaging analysis was used to identify the overlap region of the Ri B cell zones. Figure 5: Panels A, D, G are stained with B220 / CD4 LN from treated mice, respectively, with LTB-R-Ig, LFA-3-Ig and TNF-R55Ig. Fluorescence images are analyzed using imaging software analysis. Panels B, E, H show staining for sialoadhesin and panels C, F, I show staining for MOMA-1.

EXAMPLE 7

Effect of LTB-R-Ig treatment on antibody response to SRBC

Balb / c mice were injected with LTB-R-Ig or human Ig or PBS as follows: Figure 6 A: mice received 6 injections as described in Figure 2, Example 3. Animals were bled on day 7 ( black bars) and on day 14 (striped bands) after immunization with SRBC. Figure 6 C. Animals _? received hybrid proteins on day -7 and day 0. SRBCs were given on day 0 and animals were bled on day 7 (black bars), day 14 (striped bands) and day 10 (white bars). Figure 6 C. Animals receiving hybrid proteins once on day 0 at the same time as SRBC immunization. Blood was collected on day 7 (black bars), on day 14 (striped bands), and on day 34 (gray bars).

The titer of SRBC-specific IgM and IgG was determined by serum analysis by hemagglutination. The titer was defined as reciprocal of the last serum dilution, in which hemagglutination was detected and represented as Log base 2 scale (1 = dilution 1/15 of serum). The results presented are averaged from 4 different animals in the standard deviation group.

Claims (31)

Patent Claims A method of altering the humoral immune response in an animal organism, comprising the step of a) administering a pharmaceutical composition comprising a therapeutically effective amount of an LTp-R blocking agent. The method of claim 1, wherein the LTp-R blocking agent is selected from the group consisting of: soluble lymphotoxin-β receptor, antibody directed against the LT-β receptor, and an antibody directed against the surface LT ligand. 3. The method of claim 1, wherein the animal is a mammal. The method of claim 3, wherein the mammal is a human. The method of claim 2, wherein the ΕΤ-β-R blocking agent comprises a soluble lymphotoxin-β S receptor having a ligand binding domain that can selectively bind to a surface LT ligand. t A method according to claim 5, characterized in that the soluble lymphotoxin-β receptor comprises a human immunoglobulin Fc domain. The method of claim 2, wherein the ΕΤβ-R blocking agent comprises a monoclonal antibody directed against the LT-β receptor. A method according to claim 7, characterized in that the composition is introduced in an amount sufficient to cover LT-β receptor-positive cells for 1 to about 14 days. The method of claim 7, wherein the ΕΤ-β-R blocking agent comprises the anti-human ίΤ-β-R mAb BDA8. 10. The method of claim 2, wherein the LT-β-R blocking agent comprises a monoclonal antibody directed against a surface LT ligand. A method according to claim 10, characterized in that the composition is introduced in an amount sufficient to cover surface LT ligand-positive cells for 1 to 14 days. 12. The method of claim 10, wherein the antibody is directed against a LT ligand subunit. 13. The method of claim 12, wherein the LT-β-R blocking agent comprises an anti-human LT-β mAb B9. The method of claim 10, wherein the LT-β-R blocking agent comprises a monoclonal antibody directed against rat surface LT ligand. The method of claim 1, comprising a pharmaceutically acceptable carrier or adjuvant. 16. The method of claim 1, wherein the humoral immune response is suppressed. The method of claim 1, wherein said patient is infected with a human immunodeficiency virus. A method of treating, preventing or eliminating a human immunodeficiency virus in a mammal, comprising the step of administering a pharmaceutical composition comprising a therapeutically effective amount of an LT-β-R blocking agent and a pharmaceutically effective carrier. 19. The method of claim 18, wherein the LT-P-R blocking agent is selected from the group consisting of soluble lymphotoxin β-R and an antibody directed against LT-P-R and an antibody directed against a surface LT ligand. 20. The method of claim 19, wherein the blocking agent comprises soluble lymphotoxin-3-I containing a ligand binding domain that can selectively bind to a surface LT ligand. 21. The method of claim 20, wherein the soluble receptor comprises a human immunoglobulin Fc domain. 22. The method of claim 18, wherein the LT-3-R blocking agent comprises a monoclonal antibody directed against LT-3-R. 23. The method of claim 22, wherein the blocking agent comprises the anti-human LT-3-R mAb BDA8. A method according to claim 18, wherein the blocking agent comprises a monoclonal antibody directed against a surface LT ligand. The method of claim 18, further comprising the simultaneous administration of an additional anti-viral agent. A pharmaceutical composition for the treatment of graft rejection, comprising a therapeutically effective amount of a blocking LT-3-R agent and a therapeutically effective amount of a blocking CD40L. A composition according to claim 26, wherein the LT-3-R blocking agent is LT-β-R / IgG and the CD40L blocking agent is an anti-CD40L compound. A pharmaceutical composition for the treatment of spin or HIV, which comprises AZT, a protease inhibitor and a blocking agent of LT-3-R. A composition according to claim 28, wherein the blocking agent is an LT-β-R / IgG hybrid. The composition of claim 27, wherein the aHTH-CD40L compound is a monoclonal antibody. A composition according to claim 30, wherein the antibody is 5c8.